BE1025210B1 - IMMUNOGENIC CONJUGATES AND THEIR USE - Google Patents

Abstract

The technology provided is in the field of conjugation of native non-detergent native membrane vesicles (nOMV) to antigens to form nOMV-antigen conjugates, which are particularly useful for immunogenic compositions and immunization; methods for the preparation and use of such conjugates are also provided.

Description

IMMUNOGENIC CONJUGATES AND THEIR USE

This invention is in the field of conjugation of “native” external membrane vesicles (nOMV), not extracted by a detergent, with antigens, to form nOMV-antigen conjugates, which are particularly useful for immunization.

1 ¹ art background

Conjugation of antigens to carriers is an established procedure to improve immunogenicity, especially for saccharides. For example, bacterial capsular saccharides are antigens naturally independent of T cells that give rise to an immune response that lacks several important properties. Conjugation to a support moiety converts these saccharides into T-cell dependent antigens which can then produce an immunological memory effect, and also trigger effective immune responses in young children.

A known source of protein support in such conjugates is the membrane protein complex

BE2017 / 5855 external (OMPC) of N. meningitidis serogroup B (for example, see document EP-0467714, Merck & co.), Which has been included as a carrier in approved vaccines based on H. conjugates influenzae B. OMPC has also been used as a carrier in protein conjugates. According to the prior art, OMPC is conjugated to an antigen via a protein residue, which can be activated or chemically modified in order to more efficiently effect conjugation with the chosen antigen.

Wu et al. (PNAS USA 2006; 103 (48): 18243-18248) report that conjugation of Pfs25H (a human protein blocking malaria transmission) to OMPC has resulted in a Pfs25H-OMPC conjugate vaccine which has been> 1000 times more powerful in the production of anti-Pfs25H reactivity by the ELISA technique in mice compared to a similar dose of Pfs25H alone. Conjugation of OMPC to the protein Pfs25H can be obtained by reacting the Pfs25H activated by a maleimide with thiolated outer membrane proteins within the OMPC (for a general reference, see, for example, document WO 2006 / 124712), as shown in diagram 1.

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(OMPC i ί Pf $ 25H J 1 i Q / y / i \ ƒ f ....... X. \ 1 ... Λ ..... J. K 7 '> »<} ··· PB25 s X i l 3 i X X /

*

X f / I \ —f $ Sfe ”~? pfs2SH $ 'L ^ν Λ _δ ^Λ

Diagram 1

Even though the process may represent a valid synthetic route, the vesicles under consideration may be difficult to obtain in pure form, and they are usually collected by laborious processes. In addition, the connection with the selected antigen requires the presence and activation of an appropriate vesicle protein, thus posing an additional problem in light of the use of detergents or chemical agents during isolation of the vesicles. , which can change the composition of surface proteins. Therefore, there is always the need to provide new conjugates useful as immunogenic compounds which overcome the problems of the prior art, and which can be obtained by a simple and practical procedure.

The Applicant has now discovered that when nOMVs are connected to antigens selected by

BE2017 / 5855 via saccharide fractions, the conjugates thus obtained are endowed with remarkable immunogenicity and can be obtained by a reliable and practical process, as described below in more detail.

Summary of the invention

In a first aspect, the invention relates to an immunogenic nOMV-antigen conjugate, comprising a native external membrane vesicle (nOMV) obtained by a detergent-free process, comprising at least one fraction of native surface saccharide connected to at least a selected foreign antigen.

In another aspect, the invention relates to a process for preparing said conjugate, comprising the following steps:

i) the activation of at least a fraction of nOMV saccharide, generally linked to the surface of nOMV, and ii) the connection of the activated saccharide thus obtained to at least one selected antigen.

According to a preferred embodiment, the saccharides linked to the surface of the nOMVs are activated by oxidation, and then connected with the selected antigens, more preferably under conditions of reductive amination.

In another aspect, the invention also relates to the above conjugate for use as a medicament, particularly as an immunogenic compound, or for the preparation of an immunogenic composition or a vaccine.

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In yet another aspect, the invention relates to an immunogenic composition or a vaccine, comprising the conjugate indicated above and at least one pharmaceutically acceptable carrier or adjuvant; and a method for producing an immune response in a vertebrate, comprising administering said composition or said vaccine.

In another aspect, the invention also relates to the use of nOMV for the preparation of immunogenic conjugates.

Brief description of the drawings

Figure 1 shows the structures of -OAg for S. sonnei.

FIG. 2 shows the anti-Vi (FIG. 2A) and anti-OAg (FIG. 2B) IgG titers after immunization with the fragmented Vi saccharide (fVi) conjugated to nOMVs of S. typhimurium compared to fVi physically mixed with said nOMVs or combined with the more traditional support, CRM197 (formulated with Alhydrogel). 5 week old female CD1 mice (8 per group) were immunized subcutaneously on days 0 and 28 with 1 µg Vi / dose. Titers were measured on days 0, 14, 28 and 42.

FIG. 3 represents the antiCTF1232 IgG titers after immunization with the CTF1232 polypeptide conjugated to various supports (formulated with Alhydrogel). Female CD1 mice, 5 weeks old (8 per group) were vaccinated intranasally with 30 μΐ of vaccine. (15 μΐ per nostril) on the days of the study 0, 21

BE2017 / 5855 and 38. The sera were collected on days 0, 14, 35 and 52. The dose was 0.5 pg of CTF1232.

FIG. 4 represents the anti-Pfs25 IgG titers after immunization with the Pfs25 polypeptide conjugated to nOMVs of S. typhimurium 1418 AtolR compared to Pfs25 alone or physically mixed with said nOMVs. 5 week old female CD1 mice (8 per group) were immunized subcutaneously on days 0 and 28 with 0.1 µg Pfs25 / dose (Figure 4A, with Alhydrogel) or 1 µg Pfs25 / dose (Figure 4B, without Alhydrogel). The titers were measured on days 0, 14, 28 and 42. FIG. 4C represents the IgG antiOAg titers using 2 and 0.1 μg of Pfs25 / dose, and corresponding to 10 and 0.5 μg of nOMV / dose , respectively (formulated with Alhydrogel). Again, female CD1 mice, 5 weeks old (8 per group) were immunized subcutaneously on days 0 and 28.

FIG. 5 represents the anti-RO6C IgG titers (FIG. 5A) and the anti-OAg IgG titers (FIG. 5B) after immunization with RO6C alone, or conjugated to nOMV vesicles of S. typhimurium 1418 AtolR (formulated with Alhydrogel ). A recycled conjugate was prepared by recycling unconjugated RO6C from the first conjugation batch. 5 week old female CD1 mice (8 per group) were immunized subcutaneously on days 0 and 28, and doses of 1, 4 and 20 µg RO6C were used. For conjugates, the corresponding doses of nOMV were 13 pg, 52 pg and 258 pg, respectively (4 pg of RO6C and 32 pg of nOMV

BE2017 / 5855 for recycled conjugate). IgG titers were measured on days 0, 14, 28 and 42.

Figure 6a - Anti-Pfs25 IgG response induced in mice (200 μΐ per dose injected in SC on days 0 and 28, samples on days 0, 14, 27 and 42) by nOMV conjugates of the invention produced by conjugation nOMV of S. typhimurium with the Pfs25 antigen, with or without neutralization reaction, according to the embodiments of

1'invention.

Figure 6b - Anti-OAg response induced in mice (200 μΐ per dose injected in SC on days 0 and 28, samples on days 0, 14, 27 and 42) with nOMV conjugates of the invention produced by conjugation of nOMV of S. typhimurium with the Pfs25 antigen, with or without a neutralization reaction, according to the embodiments of the invention.

Detailed description of the invention

To facilitate understanding of the present invention, a number of terms and phrases are defined below. Synonyms or variations recognized in the art of the following terms and phrases (including past tenses, present tenses, etc.), even if not specifically described, are contemplated

As used in the present disclosure and the claims, the singular forms "one", "one", "the" and "the" include the plural forms unless the context clearly indicates otherwise; that is, "one" means "one or more" unless otherwise indicated.

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The terms "approximately" or "approximately" mean roughly, around, or in the regions of. The terms "approximately" or "approximately" further mean within an acceptable range of contextual errors for the particular value determined by a person of average skill in the field, which will depend in part on how the value is measured or determined , that is, the limitations of the measurement system or the degree of precision required for a particular purpose, for example, the amount of a nutrient in a nutrient formulation. When the terms "approximately" or "approximately" are used in conjunction with a numeric range, they modify that range by extending the limits above and below the numerical values presented. For example, "between about 0.2 and 5.0 mg / ml" means that the limits of the numeric range extend below 0.2 and above 5.0 so that the particular value in question is reached the same functional result as within the range. For example, "approximately" and "approximately" may mean 1 or more than 1 standard deviation depending on practice in the art. Alternatively, "approximately" and "approximately" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% a given value.

The term "and / or" as used in a sentence such as "A and / or B" is intended to include "A and B", "A or B", "A", and "B". Similarly, the term "and / or" as used in a sentence such as "A,

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B, and / or VS " East supposed to encompass each of the modes of production following: A, B, and C ; AT, B , or VS ; AT or VS ; A or B ; B or VS ; A and C; A and B ; B and C; AT (alone) ; B (alone) ; and C (alone). Except precision contrary to all the designations "A% -B% " , "A-B -g "," A o cL B% " " AT at B% "

"A% -B", "A% to B", it is given the usual and conventional meaning. In some embodiments, these designations are synonymous.

The terms "substantially" or "sensitive" mean that the condition described or claimed works in all important aspects like the standard described. Thus, "substantially free" is meant to encompass conditions which work in all important aspects as free conditions, even if the numerical values indicate the presence of certain impurities or substances. "Sensitive" generally means a value greater than 90%, preferably greater than 95%, most preferably greater than 99%. When particular values are used in the specification and in the claims, unless otherwise indicated, the term "substantially" means with an acceptable range of errors for the particular value.

An "effective amount" means an amount sufficient to cause the referenced effect or result.

An "effective amount can be determined empirically and routinely using techniques known in connection with the stated objective.

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As used herein, "heterologous" means that the two or more referenced molecules or structures are derived from a different organism. For example, a heterologous antigen is one that is derived from an organism different from the nOMV vesicle to which it is attached. "Homologous" as used herein means that the two or more referenced molecules or structures are derived from the same organism.

As used herein, "foreign" means that the two or more referenced molecules or structures are not naturally associated with each other. For example, a selected antigen which is here supposed to be "foreign to" a surface saccharide of nOMV here means that the antigen is not naturally or innately conjugated to the surface saccharide and, therefore, is not naturally conjugated to the nOMV molecule even though the antigen and the saccharide (or the nOMV molecule) may come from the same organism. In this way, a foreign antigen is not necessarily a heterologous antigen but a heterologous antigen is a foreign antigen.

"Sequence identity" can be determined by the Smith-Waterman homology search algorithm as implemented in the Oxford Molecular (MPSRCH) program, using an affine gap search with the penalty parameters d gap opening = 12 and gap extension penalty = 1, but is preferably determined by the Needleman-Wunsch global alignment algorithm (see, for example, Rubin (2000) Pediatric. Clin. North Am. 47: 269-285), using the default settings (par

BE2017 / 5855 example, with a gap opening penalty = 10.0, and with a gap extension penalty = 0.5, using the score matrix EBLOSUM62). This algorithm is practically implemented in the Needle tool of the EMBOSS software package. When the request relates to the sequence identity with a particular SEQ ID, the identity is assumed to be calculated over the entire length of that SEQ ID.

The term "w / w in%" indicates the percentage by weight of a given component, relative to a different component or relative to the total content of a composition, as indicated.

Similarly, the term "% v / v" indicates the percentage by volume of a given component, relative to a different component or relative to the total content of a composition, as indicated.

The term “-OAg” (O antigen) is used within the present invention to indicate an antigen functionality present in lipopolysaccharides (LPS) or lipooligosaccharides (LOS) on the surface of the nOMV considered, useful for conjugation with a correct antigen (generally indicated by Ag) according to the invention. In more detail, LPS are generally formed from three different parts, known as: lipid A (responsible for the toxicity of LPS), the oligosaccharide nucleus and the -OAg chain, a repeating polymer of glycans and the main contributor to the serological specificity of bacteria.

The term "alkyl or alkenyl to C linear or branched _x" includes within its meaning a

BE2017 / 5855 linear or branched saturated or unsaturated linear or branched alkyl or alkenyl group containing 1 to x carbon atoms. For example, the term bivalent C1 to C10 alkyl or alkenyl group includes in its meaning a bivalent saturated or unsaturated alkyl or alkenyl group having 1 to 10 carbon atoms such as methyl, ethyl, vinyl, allyl and the like.

As used herein, the term "saccharide (or sugar) moiety" includes in its meaning monosaccharides, as well as polysaccharide units. It should be understood that the saccharide fractions can exist in open and closed form (cycle) and that, when closed forms are presented here in the structural formulas, the open forms are also encompassed by the invention. Similarly, it should be understood that the saccharide fractions can exist in forms of pyranose and furanose and that, when forms of pyranose are presented here in the structural formulas, forms of furanose are also encompassed. Different anomeric forms of saccharide moieties are also included.

The term "oligosaccharide" includes in its meaning polysaccharides comprising from 3 to monosaccharide units.

Unless otherwise provided, the term "polypeptide" refers to polypeptides of any length capable of acting as the chosen antigen. The amino acid polymer forming the polypeptide of the invention may be linear or branched, may include modified amino acids, and it

BE2017 / 5855 can be interrupted by non-amino acids. The term also includes an amino acid polymer that has been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. 'art. The polypeptides can appear as single chains or as associated chains.

The "weight average molecular weight" is intended to indicate the weight average molecular weight obtained by ordinary arithmetic means or the average of the molecular weights of the individual component, for example, amino acids in the case of polypeptide derivatives.

The term "polysaccharides / capsular saccharides" (PSC) indicates those saccharides that may be found in the layer that lies outside the cell envelope of bacteria, thus forming part of the outer envelope of the bacterial cell itself . PSCs are expressed on the outermost surface of a wide range of bacteria, and in some cases even in fungi.

Unless otherwise provided, the term "conjugation" indicates the connection or connection of

BE2017 / 5855 subject entities, particularly with reference to the nOMV and the selected antigenic fractions.

By "immunologically effective amount" it is meant that the administration of this amount to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. This amount may vary depending on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (for example, non-human primate, primate, etc.), the capacity of the immune system. of the individual to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the physician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall within a relatively wide range which can be determined through routine testing.

The term "nOMV" here indicates a vesicle isolated from the medium or detached from cells, and these are intact membrane vesicles not exposed to detergents or denaturing agents, that is to say, not extracted by detergent. The nOMVs of the invention present the outer membrane proteins (OMP) and the lipopolysaccharide (LPS) in their native conformation and correct orientation in the natural membrane environment, and in which the cytoplasmic components are usually lacking.

On the contrary, the term “OMV” or “dOMV” encompasses various proteoliposomal vesicles obtained by rupture of the outer membrane of a Gram negative bacterium generally by an extraction process by a

BE2017 / 5855 detergent to form vesicles from it. Complexes of outer membrane proteins (for example, Neisseria meningitidis OMPC) can be considered in such a definition, because they have a three-dimensional structure and composition similar to dOMV, and they are isolated by procedures extraction with a detergent (see, for example, documents EP 0467714, US 4,271,147, US 4,459,286 and US 4,830,852). The detergent extraction process removes LPS and phospholipids, together with immunoprotective lipoproteins. Such elimination changes the structure of native vesicles and promotes aggregation. Aggregation can lead to significant problems in terms of process development (yield, regularity of production and stability). Unlike nOMVs, characterized by a defined homogeneous distribution of sizes (generally in the range of 20 to 250 nm, measured by the dynamic DLS light scattering technique), dOMVs have an indefinite heterogeneous distribution of sizes (usually in the range of 550 to 5500 nm measured by the dynamic DLS light scattering technique) caused by detergent-induced vesicle aggregation (see for general reference, Vaccine 28, 2010, 4810). The detergent extraction process also causes contamination of the OMV-containing composition (eg, vaccines) with cytoplasmic proteins as a result of lysis of the bacterial cells.

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According to methodologies of the prior art, dOMVs and nOMVs can be analyzed and described in terms of size, shape and overall appearance of impurities or non-OMV contaminating materials (such as vesicle aggregates or residues of detergent in the case of dOMV) using transmission electron microscopy (TEM). For detailed references regarding the differences between dOMVs and nOMVs, see, for example, van de Waterbeemd (2013) J. Prot. Res. Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines; and J. Klimentova et al. Microbiological Research 170 (2015) 1-9 Methods of isolation and purification of the outer membrane vesicles from gram-negative bacteria.

As indicated above, in a first aspect, the invention relates to a conjugate comprising a selected antigen connected to a saccharide fraction present on the surface of a vesicle of native external membrane not extracted by a detergent (nOMV ). Note, the nOMVs according to the present invention are collected and isolated substantially without the use of detergents, unlike, for example, dOMVs of the prior art obtained by means of an extraction with deoxycholate or in using zwitterionic detergents such as Empigen BB (see, for example, US 4,707,543) or the like. On the contrary, it should be emphasized that a detergent extraction step may be undesirable in the present invention, for a whole series of reasons, including the fact that a

BE2017 / 5855 detergent will reduce the amount of lipopolysaccharide (LPS) / lipooligosaccharide (LOS) present on the vesicle, which can be really useful for conjugation with the chosen antigen as described below.

In more detail, nOMVs are naturally occurring membrane vesicles which form spontaneously during bacterial growth and which are released into the culture medium. They can be obtained, for example, by culture of bacteria in a broth culture medium, by separating whole cells from the smaller nOMVs in the broth culture medium (for example, by filtration or by low speed centrifugation to aggregate only cells and not smaller vesicles), and then collecting nOMVs from cell-depleted medium (for example, by filtration, by differential precipitation or aggregation, by high speed centrifugation to aggregate the vesicles). Strains for use in the production of nOMV can generally be chosen based on the amount of nOMV produced in culture. These nOMV are characterized by the fact that they are collected and isolated following a procedure devoid of detergent. Preferably, the present nOMVs are released into the fermentation broth and are purified using centrifugation and a subsequent filtration step (for general reference, see, for example, Clin

Immunol vaccine.

still preferred, the present nOMV are released in the fermentation broth and are purified in

BE2017 / 5855 using the following two consecutive stages of tangential flow filtration (FFT): (i) microfiltration in which the culture supernatant containing the nOMV is separated from the bacteria, and (ii) an ultrafiltration in which the nOMV are separated soluble proteins (for a general reference, see, for example, PLoS One. 2015; 10 (8): e0134478). The nOMVs thus obtained can then be used directly within the present invention without any additional purification / isolation step. The nOMVs presently considered have a preferred size distribution between 20 and 250 nm, measured by the dynamic DLS light scattering technique.

According to some embodiments, nOMVs are prepared from wild type bacteria or from bacteria that have been genetically engineered in general to increase immunogenicity (for example, to hyper-express immunogens), to reduce toxicity , to inhibit the synthesis of capsular saccharides, to negatively regulate the expression of immunodominant antigens, and the like. They can also be prepared from hyperburgical strains. The nOMVs of the invention can also express exogenous proteins on their surface and they can be depleted of endotoxin.

Preferably, the nOMVs to be used in the present invention are produced from genetically modified bacterial strains which are mutated to enhance the production of vesicles, and possibly also to eliminate or modify

BE2017 / 5855 antigens (e.g. lipid A) and / or to overexpress homologous antigens or antigens from other organisms. Said preferred nOMVs are also known as generalized modules of membrane antigens (GMMA) as described, for example, in PLoS One. 2015; 10 (8): e0134478.

The amplified spontaneous production of vesicles can be obtained, for example, by a targeted deletion of proteins involved in maintaining the integrity of the membrane. It has been observed that the external surface of nOMVs substantially corresponds to the external surface of the bacteria from which they are derived, preserving the membrane antigens (including, for example, lipopolysaccharides, lipooligosaccharides and lipoproteins) in the background of the membrane. Advantageously, the nOMVs used in the invention (unlike the dOMVs extracted by a detergent) retain these components of the outer membrane in their native conformation and correct orientation, better preserving immunogenicity against the bacterial strain from which they are derived.

Generally, nOMVs for use in the present invention can be prepared from any suitable bacteria, where preferred bacteria include, but are not limited to: Neisseria (for example, especially N. meningitidis of any serogroup including A, B, C, X, Y or W135, or from a non-pathogenic Neisseria), Shigella (like S. sonnei, S. flexneri, dysenteriae or boydii), serovars of Salmonella enterica (such as paratyphi A,

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B or C, enteritidis, typhi or typhimurium), Haemophilus influenzae (e.g. H. influenzae not typeable), Vibrio cholerae, Bordetella pertussis, Mycobacterium smegmatis, Mycobacterium bovis BCG, Escherichia coli, Bacteroides (including Porphyromonas aer) Helicobacter pylori, Brucella melitensis Campylobacter jejuni, Actinobacillus actinomycetemcomitans, Xenorhabdus nematophilus, Moraxella catarrhalis, or Borrelia burgdorferi.

The particularly preferred bacteria are chosen from at least one of: S. sonnei, S. flexneri, the bacteria Salmonella, and the meningococcus, particularly the meningococcus of serogroup B.

Virulent Shigella strains have a 220 kb plasmid that mediates virulence properties. This "virulence plasmid" has been shown to encode genes for several aspects of Shigella virulence, including adhesins for target epithelial cells, invasive plasmid antigens, virF, virG, and the like. A Shigella used with the invention may or may not have a virulence plasmid. The absence of the plasmid can stabilize the strain during industrial culture, attenuate the strain by eliminating virulence factors (thereby increasing the safety of manufacture), avoid the presence of the enterotoxin ShET2 (coded by the ospD3 gene or sen on the plasmid), and avoid the presence of msbB2 which is a second copy of the msbB gene responsible for the acylation of lipid A. The absence of the virulence plasmid can also disturb the lipopolysaccharide. However, the genes for

BE2017 / 5855 biosynthesis for 1'-OAg should preferably be conserved, either by the maintenance of a mutated virulence plasmid, or by inclusion in another plasmid or by cloning in the bacterial chromosome

As far as the Salmonella bacterium is concerned, a particularly preferred strain is chosen from: Salmonella typhimurium, Salmonella enteritidis and Salmonella paratyphi A.

The nOMVs of the Meningococcus bacteria are also preferred. Such vesicles can be prepared from any meningococcal strain. The vesicles are preferably prepared from a strain of serogroup B, but it is also preferred to

to prepare at from serogroup s others that B, as One of: AT , vs, W135 or Y, depending of the known procedures in art. The strain may be of all serotype (through example, 1 2a, 2b, 4 , 14, 15, 16 etc. ) , all sero

subtype (for example, PI.4), and any immunotype (for example, L1; L2; L3; L3.7; L3.7.9; L10; etc.). The meningococci can be of any suitable line, including hyper-invasive and hypervirulent lines, preferably any one of the following seven hypervirulent lines: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; group A4; line 3. Most preferably, the OMVs are prepared from the strain NZ98 / 254, or another strain with the sero-subtype of PorA PI.4.

In another embodiment, the bacteria for the preparation of the nOMVs useful for the invention can be mutant strains which have been manipulated,

BE2017 / 5855 for example, to amplify the production of vesicles, to express one or more desired antigens, and / or to inactivate or modify an unwanted gene (for example, one which codes for a toxin or which codes for an enzyme involved in production of a toxic product, such as an endotoxin).

In this direction, other nOMVs for the invention are produced by a Salmonella bacterium, particularly an S. typhimurium (also known as Salmonella enterica serovar typhimurium) which does not express a functional TolR protein.

When the vesicles are prepared from E. coli, Shigella or Salmonella, the bacteria cannot express more than 4 of the proteins TolA, TolB, TolQ, TolR and Pal. Thus, at least one protein from the system of five natural Tol-Pal proteins may be absent, resulting in a bacterium which, during growth in the culture medium, releases greater amounts of outer membrane vesicles in the medium compared to the same bacteria expressing the 5 Tol-Pal proteins. Preferably TolR is not expressed, but the other four proteins can be expressed (i.e., an ATolR strain).

In preferred embodiments, at least one of the five Tol-Pal proteins in E. coli, Shigella or Salmonella is eliminated, for example, by deletion or inactivation of the gene encoding the protein. Thus, the bacteria can express 0, 1, 2, 3 or 4 of the proteins TolA, TolB, TolQ, TolR and Pal. Elimination of one of the five proteins may suffice, in which case the bacteria expresses only 4 of these proteins. Preferably, the

BE2017 / 5855 TolR protein is eliminated, for example, by inactivation of the tolR gene of a starting strain. Thus, a preferred bacterium can be tolA + tolB + tolQ + TolRPal +.

In some embodiments, the bacteria express the five Tol-Pal proteins, but at least one is mutated to cause hyper-budding. For example, the protein TolA, TolQ, TolR and / or Pal can be mutated in such a way that the protein retains its membrane localization but its interactions with the other members of the Tol-Pal system are disturbed. The bacteria will thus keep TolA, TolQ and TolR as transmembrane proteins in the internal membrane, and the Pal protein as lipoprotein facing the periplasm in the external membrane, but at least one of the proteins TolA, TolQ, TolR and / or Pal is mutated and not fully functional.

In addition, other mutations may also be present, for example, to give OAg deficient strains, for example in those cases where the -OAg functionality is not expected for the desired immune response, or in these cases where 1 '-OAG may have a negative impact on immunogenicity against the heterologous antigen. In this direction, the possible mutations can be AgalU, AgalE or AwbaP in strains of E. coli, Shigella or Salmonella.

In another preferred embodiment, a meningococcus does not express a functional MltA protein. Inactivation of MltA (lytic membrane-bound transglycosylase, also known as GNA33) in the meningococcus provides

BE2017 / 5855 bacteria which spontaneously release large quantities of nOMV into the culture medium, from which they can be easily purified. For example, the vesicles can be purified using the two-step size filtration process, comprising: (i) a first filtration step in which the vesicles are separated from bacteria based on their different sizes, with the vesicles passing in the filtrate; and (ii) a second filtration step in which the vesicles are retained in the retentate.

In the present invention, it is preferred that 1 -OAg is present on nOMVs because we have observed (for example, nOMVs originating from Salmonella and Shigella) that, the presence of 1'-OAg on the surface of said nOMVs is advantageous in providing a bivalent vaccine, since 1'-OAg can act as a protective antigen. Some preferred strains have a less toxic penta- or tetra-acylated LPS, which includes fixed 1'-OAg, after the mutation of msbB, htrB, ddg and / or PagP (see, for example, Rossi O et al., Clin Vaccine Immunol. April 4, 2016; 23 (4): 304-14 and Rossi O et al., J Biol Chem. September 5, 2014; 289 (36): 24922-35.

In Neisseria, the strain preferably includes a modified fur gene. According to this embodiment, the mutant Neisseria are modified to reduce or deactivate the expression of at least one gene involved in rendering the lipid A part of the LPS toxic, in particular of the lpxll gene. In this way, the resulting nOMVs exhibit reduced toxicity compared to the strain

BE2017 / 5855 wild type, since the conversion of acylated lipid A into a less acylated form.

Similarly, the preferred Neisseria mutants for the invention are modified to reduce or deactivate the expression of at least one gene involved in the synthesis or export of capsular saccharides, in particular the synX and / or ctrA genes. In this way, the resulting nOMVs can exhibit cross-protection against various serotypes, which is particularly appreciated by those skilled in the art.

In preferred embodiments, a strain may include one or more of the inactivation and / or hyper-expression mutations disclosed, for example, in Fukusawa et al. (1999), Vaccine 17: 29512958. For example, following the advice and the nomenclature indicated therein, the genes useful for negative regulation and / or inactivation include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB / MsbB, LbpA, LbpB, LpxK, Opa, Ope, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB / MsbB, LbpA, LbpB, LpxK, Opa, Ope, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE,

LbpA, LpbB, Opa, Ope, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and / or TbpB; or (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, SynX and / or SynC.

As mentioned above, the present conjugates are obtained by connecting at least one surface saccharide fraction of nOMV (preferably included in the related 1'-OAg) to one or more antigens

BE2017 / 5855 foreigners chosen, that is to say who are not part of the gallbladder.

As far as the saccharide fraction of nOMV is concerned, it should be noted that it can be part of the -OAg functionality present on the surface of nOMV (for example, in LPS or LOS), or it can be present within from a different part of the surface of the nOMV, for example a PSC, as described below in detail. Advantageously, any correct antigen can be conjugated to nOMV to obtain the nOMV-antigen conjugates of the invention, preferably in the form of a (poly) saccharide or a polypeptide. In all cases, the connection of one or more selected antigens produces an immunogenic conjugate which can produce an immune response which recognizes the antigenic art, and which also recognizes one or more components in; practical and useful for the multivalent. The antigens present conjugated to a high enough to be administered to a host, a nOMV, so in preparation for a vaccine will be included in the concentration which is: lencher, when they are immune response which recognizes this antigen. In addition, the immune response is preferably protective against the pathogen from which the antigen was derived, even more preferably against one of the pathogens listed below.

In one embodiment of the invention, the nOMV is conjugated to at least one homologous antigen, that is to say derived from the same organism from which the nOMV are derived. In a still preferred embodiment,

BE2017 / 5855 the antigen chosen is a heterologous antigen, that is to say derived from an organism different from the organism from which the nOMVs are derived.

In all cases, the antigens are generally chosen from any immunogenic polypeptides, that is to say polypeptides capable of triggering an immune response when they are administered to a subject. The polypeptides used with the invention will comprise an amino acid having a residue, or a side chain, with a functional group suitable for conjugation, preferably an amino or thiol group, even more preferably of general formula: -NH2 or - SH. These residues can be naturally present in an antigen, or they can be introduced artificially for the purposes of conjugation. Preferred amino acid residues include, but are not limited to: arginine, lysine, asparagine, glutamine, cysteine and histidine. The most preferred amino acid residue for conjugation is lysine. In fact, its side chain -NH2 can react with an oxidized -OH group activated from a saccharide fraction of nOMV and can react with the aldehyde group thus obtained by reductive amination, according to the method disclosed here in detail.

The polypeptide antigens are preferably prepared in a substantially pure or substantially isolated form (i.e., substantially free of other polypeptides). They can be prepared by various means, for example, by chemical synthesis (at least in part), by digestion of longer polypeptides using proteases, by

BE2017 / 5855 translation from RNA, by purification from a cell culture (for example, from a recombinant expression or from the native culture), and the like. Recombinant expression in a host of E. coli is a useful route of expression. Polypeptide antigens can take various forms (eg, native, fusions, glycosylated, unglycosylated, lipid, disulfide bridges and the like).

The polypeptide antigens used with the invention have a preferred weight average molecular weight of at least 1 kDa, more preferably at least 3.5 kDa, even more preferably from 10 to 80 kDa. Even more preferably, the weight average molecular weight is from 15 to 75 kDa.

Other preferred polypeptide antigens for conjugation to nOMVs according to the present invention include an epitope from a fungal, bacterial, protozoan or viral polypeptide. Preferred protozoan polypeptides are from a Plasmodium (such as P. falciparum, P. vivax, P. ovale). The particularly preferred bacterial polypeptides are chosen from: E. coli, N. meningitidis, and streptococci (such as S. agalactiae, S. pneumoniae, S. pyogenes).

E. polypeptide antigens. preferred coli include CTF1232, 405 and 3526. As preferred nonlimiting examples, nOMVs from Shigella or Salmonella can be conjugated to CTF1232, according to the present invention, to produce a bivalent vaccine covering both enterotoxigenic E. coli ( ETEC) and Shigella / Salmonella.

BE2017 / 5855

In one embodiment, the N. meningitidis polypeptides under consideration are capable, when administered to a mammal, of triggering an antibody response which is bactericidal against meningococcus. Preferred N. meningitidis polypeptides for use with the invention are selected from at least one of: NHBA, NadA, NsPA, NhhA, App and fHbp, as detailed below.

NHBA antigen

The NHBA antigen was included in the published genomic sequence for the meningococcal strain B serogroup MC58 as gene NMB2132 (GenBank accession number GI: 7227388; SEQ ID NO: 2 here). The NHBA antigen sequences of many strains have been published since then. Various immunogenic fragments of the NHBA antigen have also been reported. Preferred NHBA antigens for use with the invention include an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more)

with SEQ ID NO: 2; and or (b) comprising a fragment at least "N" acids amino consecutive of SEQ ID NO ; 2, in which "N" is worth 7 or more (through example, 8 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50 60, 70, 80, , 90, 100, 150, . 200, 250 or more) . The

preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 2. The most useful NHBA antigens of the invention can trigger antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the sequence

BE2017 / 5855 of amino acids SEQ ID NO: 2. The NHBA antigens advantageous for use with the invention can trigger bactericidal anti-meningococcal antibodies after administration to a subject.

NadA antigen

The NadA antigen was included in the published genomic sequence for the meningococcal serogroup B strain MC58 (see, for example, Tettelin et al. (2000) Science 287: 1809-1815) as the gene NMB1994 (number accession GenBank GI: 7227256; SEQ ID NO: 3 here). The NadA gene sequences of many strains have been published since then, and the activity of the protein as neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported. Preferred NadA antigens for use with the invention include a

amino acid sequence: (at) with 50% or more identity (e.g. 60 O Gold 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 o θ r 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 3; and or (b) comprising a fragment of at least "n" acids consecutive amines of SEQ ID NC ': 3, in which " not " is 7 or more (for example, 8 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 , 250

or more) . The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 3. The most preferred NadA antigens of the invention can trigger antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 3. NadA antigens advantageous for use with

BE2017 / 5855

The invention can trigger bactericidal meningococcal antibodies after administration to a subject. SEQ ID NO: 7 is one such fragment.

NspA antigen

The NspA antigen was included in the published genomic sequence for the meningococcal strain B serogroup MC58 (see, for example, Tettelin et al. (2000) Science 287: 1809-1815) as the gene NMB0663 (number accession GenBank GI: 7225888; SEQ ID NO: 4 here). The NspA antigen sequences of many strains have been published since then. Various immunogenic fragments of NspA have also been reported. Preferred NspA antigens for use with the invention include an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 4; and / or (b) comprising a fragment of at least “n” consecutive amino acids of SEQ ID NO: 4, in which “n” is 7 or more (for example, 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 4. The most preferred NspA antigens of the invention can trigger antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO

4. Advantageous NspA antigens for use with the invention can trigger bactericidal meningococcal antibodies after administration to a subject.

BE2017 / 5855

NhhA antigen

The NhhA antigen was included in the published genomic sequence for the meningococcal strain B serogroup MC58 (see, for example, Tettelin et al. (2000) Science 287: 1809-1815) as the antigen NMB0992 (number d 'accession GenBank GI: 7226232; SEQ ID NO: 5 here). The NhhA antigen sequences of many strains have been published since then, for example, WO 00/66741 and WO 01/55182, and various immunogenic fragments of NhhA have been reported. He is also known as Hsf. Preferred NhhA antigens for use with the invention include a

sequence acids amines: (a) with 50% or more identity (for example, 60 %, 65 O θ r 70 O θ r 75% , 80%, 85%, 90 %, 91%, 92%, 93 %, 94 o θ r 95 o θ r 96% , 97%, 98%, 99 %, 99.5% or more) with SEQ ID NO : 5 ; and or (b) comprising a fragment from to less " not " acids

amino consecutive from SEQ ID NO : 5 , in which " not " worth 7 or more (through example, 8 10 12, 14, 16, 18, 20 25, 30, 35, 40, 50 60, 70, 80, 90 100, 150, 200, 250

or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 5. The most preferred NhhA antigens of the invention can trigger antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 5. NhhA antigens advantageous for use with the invention can trigger bactericidal anti meningococcal antibodies after administration to a subject.

BE2017 / 5855

Antigen App

The App antigen was included in the published genomic sequence for the serogroup B meningococcal strain MC58 (see, for example, Tettelin et al. (2000) Science 287: 1809-1815) as an antigen

NMB1985 (GenBank GI accession number: 7227246;

SEQ ID NO: 6 here). The App antigen sequences of many strains have been published since then. Various immunogenic fragments of App have also been reported. Preferred App antigens for use with the invention include an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 6; and / or (b) comprising a fragment of at least “n” consecutive amino acids of SEQ ID NO: 6, in which “n” is 7 or more (for example, 8, 10, 12, 14, 16, 18 , 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 6. The most preferred NhhA antigens of the invention can trigger antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO

6. App antigens advantageous for use with the invention can trigger bactericidal meningococcal antibodies after administration to a subject.

FHbp antigen

The factor H binding protein exists in the form of three variants (vl, v2 and v3), and the invention

BE2017 / 5855 can use any of these as a preferred embodiment.

A v1 of fHbp preferably comprises (a) an amino acid sequence which has at least k '% identity with SEQ ID NO: 8, and / or (b) a fragment of SEQ ID NO: 8. k' refers to a percentage identity and can be defined as any number from 1 to 100. With reference to amino acid or nucleic acid sequences, the identity used in the application generally starts as low as 40% with specific references to higher percentages, i.e., 70%, 75%, 80%, etc.

The fragment will preferably comprise at least one epitope derived from SEQ ID NO: 8. Preferably, the v1 of fHbp can trigger antibodies which are bactericidal against the v1 strains, for example against the MC58 strain (available from ATCC in as "BAA-335").

An fHbp v2 preferably comprises (a) an amino acid sequence which has at least k '% identity with SEQ ID NO: 1, and / or (b) a fragment of SEQ ID NO: 1. Information concerning k 'and the fragments are given above. The fragment will preferably comprise at least one epitope derived from SEQ ID NO: 1. Preferably, the v2 of fHbp can trigger antibodies which are bactericidal against strains v2, for example, against the strain M2091 (ATCC 13091).

An fHbp v3 preferably comprises (a) an amino acid sequence which has at least k '% identity with SEQ ID NO: 9, and / or (b) a fragment of SEQ ID NO: 9. Information concerning k 'and the fragments are given above. The fragment will include

BE2017 / 5855 preferably at least one epitope derived from SEQ ID NO: 9. Preferably, the vH of fHbp can trigger antibodies which are bactericidal against strains v3, for example, against the strain M01-240355.

The group A Streptococcus (GAS), group B Streptococcus (GBS) and Pneumococcus antigens are also equally preferred. As nonlimiting examples, the antigens GAS25 (Slo), GAS40 (SpyAD) and GAS57 (SpyCEP) can be incorporated into conjugates in accordance with certain embodiments of the invention.

Plasmodium antigens are also preferred. These can come from any suitable species, where the preferred species are chosen from: P. falciparum, P. vivax and P. ovale.

Yet another preferred antigen is Pfs25 (SEQ ID NO: 10), which is an antigen of the sexual stage of P. falciparum expressed on the surface of the zygote and akinetic forms of the parasite. Another preferred antigen is Pfs48 / 45, which is a candidate vaccine that blocks transmission. Recently, the C-terminal fragment of 10 cysteines (10C) from Pfs48 / 45, containing three epitopes known for antibodies blocking transmission, was produced in the form of a chimera with the N-terminal part of GLURP (RO) , the protein rich in antigen glutamate from the asexual blood stage. The resulting fusion protein (RO10C) triggered high levels of transmission blocking antibodies in rodents (see, Theisen et al. (2014) Vaccine 32: 2623-2630). Shing et al. (2015) Vaccine 33: 1981-1986 describe a chimera containing truncated fragments of 6C, which

BE2017 / 5855 increases the yield of the correctly folded conformère. The RO6C construct was able to trigger a high titer of transmission blocking antibodies in rats RO6C (SEQ ID NO: 11) is a preferred antigen which can be conjugated according to the present invention.

Another preferred antigen is the circumsporozoite protein (CSP; SEQ ID NO: 12).

Shorter peptides from CSP can also be conjugated according to the present invention. For example, the 12 amino acid peptide (NANPH (SEQ ID NO: 13) derived from CSP can be used according to preferred embodiments.

In yet another preferred embodiment, the antigens are a species of saccharide. The invention is in fact also suitable for conjugating one or more saccharide antigens to nOMVs, whereby the saccharides can be used in their natural full-length form. As a variant, a fraction of a particular size can also be advantageously chosen. Thus, saccharides can be fragmented from their natural length, and optionally a size fraction of these fragments can be used. Even further, saccharides are not limited to saccharides purified from natural sources and synthetic or semi-synthetic saccharides can be used instead.

Preferred saccharide antigens are bacterial capsular saccharides (PSCs). These include, but are not limited to, the capsular saccharides selected from at least one of: Haemophilus

BE2017 / 5855

in fluenzae of type B; Neisseria meningitidis of serogroups AT, C, W135, X and Y; Streptococcus pneumoniae of the serotypes 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A , 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,

22F, 23F, and 33F; Salmonella including Salmonella enterica serovar typhi Vi, either full length or fragmented (indicated by fVi); Streptococcus agalactiae of serotypes la, Ib, and III; Streptococcus pyogenes, Shigella sp., Group A and B Streptococcus (GAS and GBS respectively). The more preferred saccharide antigens are from Neisseria meningitidis of serogroups A and C. Thus, according to one embodiment of the invention, the nOMV is a GMMA derived from MenB, and the antigen chosen is a capsular saccharide originating from MenA or MenC . In a still further embodiment, the invention relates to a GMMA originating from MenB conjugated to a capsular saccharide originating from the MenA antigen via a polysaccharide residue, in which said GMMA is also conjugated to a MenC antigen via a different polysaccharide residue, thereby obtaining a double functionalized GMMA vesicle, as described below in more detail.

In all cases, and as mentioned above, the antigens chosen can be conjugated to nOMVs derived from the same bacterial strain and even from a different bacterial strain, thus providing a multivalent conjugate. In this regard, in a more preferred embodiment of the invention, the nOMV and the saccharide antigen are derived from different bacterial strains.

BE2017 / 5855

Other preferred saccharide antigens are ß-glucans, which are particularly useful for protection against C. albicans (for general reference, see Sandlin et al. (1995) Infect. Immun., 63: 229-37).

Other preferred saccharide antigens are poly-rhamnose oligosaccharides for protection against group A Streptococcus (GAS). The native GAS saccharide has a poly-rhamnose backbone substituted with NAcGlcN. Synthetic polyrhamnose oligosaccharides, or oligomers with the structure of the native GAS saccharide, can be conjugated to nOMVs according to the invention.

According to a preferred embodiment, the nOMV conjugates of the invention comprise a surface saccharide fraction of nOMV directly connected to a chosen antigen, where direct connection can be obtained by activation of the saccharide fraction followed by a direct reaction ( for example, via a reductive amination) with the selected antigen, as illustrated in this example 4. In an equally preferred embodiment, the nOMV is connected to the selected antigen indirectly, that is to say, via a linker fraction, as described here in more detail and as illustrated in Example 3.

The conjugates of the invention are immunogenic, as demonstrated by studies in mice and supported by the experimental part included here. Advantageously, apart from being capable of inducing an immune response against the conjugated antigen, the conjugates of the invention are also capable

BE2017 / 5855 to induce an immune response against the nOMV component, thus being good candidates for the preparation of a multivalent immunogenic composition thereof. In fact, it has surprisingly been observed that the conjugation of the antigens chosen via the saccharide fraction present on the surface of nOMVs does not have a negative impact on the ability of nOMVs to induce their own immune response, unlike dOMV. Therefore, the conjugates of the invention may be useful, for example, as a bivalent immunogenic agent, suitable for the preparation of vaccines, with nOMV and the heterologous conjugate antigen both having good immunogenicity. In addition, it has been advantageously discovered that the conjugates of the invention induce a high response in specific anti-antigen IgG in mice, without any impact on the response of anti-OAg IgG, as it is supported for example in the present Examples 5 and 6. The conjugates of the invention offer several other advantages compared to the non-conjugated antigens, as is presented for example in Examples 3 to 6.

As previously presented, in another aspect, the invention relates to a process for the preparation of the conjugates described above, comprising a first step of activation of a surface saccharide fraction of nOMVs, and a second step of connection of the activated vesicle thus obtained to at least one selected antigen, possibly via a bivalent linker.

BE2017 / 5855

According to the present method, at least one saccharide fraction on a nOMV is conjugated to a selected antigen (as described above) to form a conjugate of the invention. According to a different embodiment, two or more saccharide fractions are conjugated to two or more different selected antigens, thus providing a nOMV derivative conjugated with two or more different antigens, which is particularly suitable for the preparation of polyvalent immunogenic compositions. As indicated above, the connection step generally involves activation of the surface saccharide fraction of the nOMVs and / or of the chosen antigen. Similarly, the connection step may involve the introduction of a linker between the saccharide fraction of the nOMVs and the selected antigen, as detailed below. Thus, in one embodiment, the method of the invention comprises the following steps: (i) activation of a saccharide fraction on the surface of the nOMVs; and (ii) direct connection of the activated fraction with a selected antigen, to obtain the nOMV conjugates of the invention

As an alternative embodiment, the method of the invention comprises the following steps: (i) activating a saccharide fraction on the surface of nOMVs; (ii) connecting the activated fraction to a bivalent linker group to form a vesicle-linker conjugate; and (iii) connecting a selected antigen to the vesicle-linker conjugate to form the nOMV conjugates of the invention.

BE2017 / 5855

As another alternative embodiment, the method of the invention comprises the following steps: (i) activation of a saccharide fraction on the surface of the nOMVs; (ii) connecting a selected antigen to a bivalent linker group to form an antigen-linker conjugate; and (iii) connecting the activated fraction from step (i) to the antigen-linker conjugate to form the nOMV conjugates of the invention.

As another alternative embodiment, the method of the invention comprises the following steps: (i) activation of a saccharide fraction on the surface of the nOMV; (ii) connecting the activated fraction to a bivalent linker group to form a vesicle-linker conjugate; (iii) connecting a selected antigen to a bivalent linker group to form an antigen-linker conjugate; and (iv) connecting the linker moiety from step (ii) to the antigen conjugate from step (iii) to form the nOMV conjugates of the invention.

As long as the saccharide fraction of nOMV is considered, it should be noted that it can be part of the -OAg functionality, or of the region of the nucleus naturally present on the surface of the nOMV (for example, in LPS or LOS). , or it may be present in a different part of the surface of the nOMV, for example, a PSC. In all these preferred cases, the method of the invention allows the connection of said saccharide fraction with an antigen chosen in a simple and effective manner, thus leading to the conjugates of final nOMVs of the invention, endowed with a remarkable immunogenic activity. . Advantageously,

BE2017 / 5855 the presence of 1'-OAg does not significantly interfere with the response against the selected antigen. Comparative Examples 9a-c show that when the present method is applied to dOMVs, no conjugation occurs with the antigen, thus preventing the formation of the desired vesicle-antigen conjugate.

Depending on the species from which the nOMVs are prepared, various saccharide fractions (including tetraose, pentose and hexose sugars) can be used for activation and subsequent conjugation. Preferably, lipopolysaccharides, via the -OAg part or of the nucleus region, or capsular saccharides can be used for activation and subsequent conjugation. The preferred saccharide fractions are chosen from at least one of the : glucose, galactose, fructose, mannose, ribose, abéquose, galactosamine, glucosamine, mannosamine, sialic acid, suifoquinovose, erythrose, threose, arabinose, rhamnose, sorbose, ribulose, xylose, xylulose, lyxose, tagatose or keto-deoxy-octoxy. A saccharide fraction on the nOMV is preferably activated by oxidation of a hydroxyl group of the saccharide to form a carbonylated aldehyde functionality, in the presence of a suitable oxidizing agent, such as TEMPO or a period salt. The latter is preferably chosen from a periodate or an alkali metaperiodate, more preferably NaI04. The oxidizing agent is preferably used in the form of an aqueous solution in a concentration in the range of 0.5 mM to 20 mM, preferably 3 mM to 20 mM, where the concentrations of 10 to 20 mM and 0.5 to 5 mM or

BE2017 / 5855 from 3 to 5 mM are even more preferred. Other activation reactions according to certain embodiments occur in the presence of:

cyanylation reagents such as

CDAP (for example, tetrafluoroborate

1-cyano-4-dimethylaminocarbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,

S-NHS, EDC and TSTU.

In general, when the polysaccharides are oxidized, it is not necessary to oxidize all of the available sugars. Indeed, it may be desirable to conserve at least part of the structures of natural sugars, particularly when these have a useful antigen. It should also be noted constitute the fact that, because of the particular composition and conformation of the nOMVs as detailed above, the polysaccharide fraction can be activated in a practical manner by the present process leading to the formation of an intermediate species highly reactive oxidized nOMV. In a preferred embodiment, for a given saccharide fraction of interest, the proportion of oxidized residues can be in the range of to 100 to or from 20 to 35%, while the oxidation of within structure of -OAg is particularly preferred. In this direction, it has been discovered that said areas allow effective conjugation with a minor or substantially absent impact on the structural integrity of the -OAg. In addition, it was noted that a higher degree of oxidation of nOMV corresponds to a smaller size of 1'-OAg, which means that there is

BE2017 / 5855 a major impact on the native structure of 1'-OAg and its ability to induce a specific immune response. The proportion of oxidized residues can be determined by high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD), by comparing the intact sugar residues of nOMV, just as the pH can influence the behavior. overall of the oxidation step, as for example it is indicated in the present example 2, table 2c. Thus, in a preferred embodiment, the oxidizing agent is used in excess relative to the starting nOMV, where a molar excess of 3/1 or 2/1 relative to the number of monosaccharides which can be subjected to the oxidation is particularly preferred. The oxidizing agent is preferably used in the form of an aqueous solution in a concentration in the range of 0.5 mM to 20 mM, preferably 3 mM to 20 mM, where concentrations of 10 to 20 mM and 0.5 to 5 or 3 to 5 mM are even more preferred.

The concentration of nOMV is preferably between 0.2 and 5 mg / ml.

Preferably, the pH is between 4 and 8, while values of 5 and 7 are particularly preferred. In this way, the pH can be adjusted using a buffering agent, such as acetate / phosphate and the like.

Said parameters can be fixed in a practical manner in order to obtain a preferred degree of oxidation of between 20% and 35% on the saccharide fraction subjected. This allows another effective conjugation with

B E2017 / 5855 the selected antigen, without appreciable impact on the structure of the saccharide fraction.

For example, Rha residues in an -OAg functionality can be oxidized, for example,

5 comm < 5 it is indic jué sur le diagram 2 below sou using Na 10.

Diagram 2

The oxidation step is generally carried out at room temperature (for example, from about 15 ° C to about 40 ° C), for an appropriate time, for example between 30 min and 3 h, depending, for example, on the quantity and type of nOMV considered. In all cases, it was discovered that there was no significant crosslinking and / or aggregation of the nOMVs. This is of the utmost importance also for

B E2017 / 5855 the effectiveness of the subsequent conjugation step with the selected antigen as described here in detail.

After the oxidation, the nOMVs can optionally be subjected to a reduction step, for example with NaBH 3, to stabilize the oxidized nOMVs by elimination of the CHO groups formed * The stabilized oxidized nOMVs can then be stored and / or further characterized.

Generally, after the activation step of the present process, the oxidized nOMVs obtained are isolated and purified, for example, by ultracentrifugation at 4 ®Ç at 110,000 rpm for 30 min, and then reacted with the selected antigen.

Thus, in a preferred embodiment, the (i) activation of the saccharide fraction on nOMV, preferably by oxidation;

isolation of the oxidized nOMVs thus obtained; and (ii) the connection of;

nOMVs oxidized with at least one selected antigen, even if it is 11 via a bivalent linker.

In a still preferred embodiment, the

N & 2SO3.

This is particularly advantageous because by neutralizing the oxidation reaction with

NasSOa, the one-step process

I sclement of oxidized nOMVs in step (i-bis) as illustrated in saving time, obtaining

B E2017 / 5855 thus the final conjugate in a simple and effective way. In practice, and according to the illustrated embodiment, after the activation step (i) the reaction is neutralized with a correct quantity of the alkali sulfite, and allowed to react for a correct duration (generally between 5 and 20 minutes) in order to neutralize the excess of the oxidizing agent. After that, the selected antigen is added directly to the mixture (i.e., without isolation of the oxidized nOMVs), according to step (II.), Thereby obtaining the nOMV conjugates of the invention.

As a variant, the carbonylated aldehyde group of the saccharide fraction obtained by the oxidation step can be further modified to form a correct functionality which can then be reacted with the chosen antigen Or with a place as can be the case (in this case to give a vesicular conjugate which can then be coupled to the chosen antigen).

The selected antigen is generally added in a ratio of 1./1 w / w with respect to the nOMVs used, at room temperature, for a correct duration, for example, between 2 hours and 24 hours. When the antigen is derivatized with a place, the reaction is. conveniently performed using an excess of the antigen over nOMVs, preferably in a ratio of 2/1 or more preferably 3/1 w / w, as indicated for example in the present example 3.

In a particularly preferred embodiment, the method of: the invention comprises the following steps: (i) the oxidation of a fraction

B E2017 / 5855 saccharide; and (ii) reacting the oxidized fraction with an amino group on a residue of the selected antigen. Even more preferably, said residue

of 1 a n t i g è ne lys residue chosen is an amin group  o -NH> on an identical polypeptide salt line; Ί an antigen Ç'IzO.l. 5.1 ” From pref erence the 3 coupling of fraction s a cchar idique oxidized by 1 .a nOMV with gr amino oupe, from preferably one -Nil group? free, from the front fen is obtained

by reductive examination, more preferably using KaBH3.CN ,. for example, according to a procedure known in the art. NaBHuO is used in quantities by weight (w / w) between 3 and 1/3, preferably 1 to 1, relative to the oxidized nOMV. Conveniently, NaBHsCN can be added together with the selected antigen, directly to the intermediate product of the oxidized nOMVs, as is generally illustrated in schemes 3 and 4 below, using, by way of example, a nOMV which is conjugated via an oxidized rhamnose unit to the malarial membrane proteins Pfs25 or R0.6.C, respectively.

I nOMV i ™ ------------->

i ..................... s ax nOMV <>

'\ ^s '\' nOMV

H \! <

"’ I C "; · 2

..- NH

PG25

Diagram 3

I

nöMV l · .........: ......... *

Maio., Οχ

B E2017 / 5855 nÖMV

As an alternative embodiment, the selected antigen can be modified, either by the introduction of a linker group or by conversion of a functional group on the antigen into another functional group suitable for the reaction with the saccharide moiety. activated on the nOMV, or with a linker of the vesicle-linker conjugate when used. In particular, if the antigen chosen is a saccharide, it can be modified by reaction with a site, either randomly (r), which means that the linker is introduced at multiple points along the sugar chain, or selectively (s), which means that the linker is introduced at the reducing end of the sugar chain (i.e., at a single position). In a preferred embodiment, the linker is introduced selectively at the terminal position of the selected antigen., As indicated for example in Example 3.

The selective modification of the antigen is preferably obtained by reaction with dihydrazide of adipic acid (& DH) in the presence of NaBHaCN, as it is generally represented in Scheme 5 using fVi as antigen. The random modification of the antigen is preferably obtained by activation of one or more carboxyl acid groups!

B E2017 / 5855 the antigen, for example using NHS / EDAC, and a subsequent reaction with. ADH, as it is shown in Figure 5 using fVi as the antigen. This type of conjugation reaction is illustrated in scheme 1 below, using, by way of example, a nOMV which, is conjugated via an oxidized rhamnose unit to the fVi modified to include an -NHa by reaction with DHA,

Diagram 5

NHS

........ i aa <.: fVi co »« ........... »

Diagram 6

I nOMV nOMV <<

<

.25 nOMV fVi-ADH 1 1

-------------> 'Ά

HaSHfN Aid: /

Ν! ^Ί ' ^ό ''ïlHCOKH.hCONHNHlVï

Diagram 7

An alternative embodiment of the invention relates to a method comprising the following steps: a) modification of an antigen chosen to include an amino group, preferably -NHs; b) the activation of a nOMV by oxidation of a hydroxyl group of a saccharide fraction as discussed above, and c) the

51 connection of fraction sa the modified antigen of (step é reductive amination, as it has A variation mode of re < a process for manufacturing a including the connection of a oxidized from a nOMV activated at making them that way the invention, or the antigen was ami.no group, preferably -NH

and iccharidic fraction oxidized to relates to an OMV-an t igene an antigen, modified.

nOMV conjugates modified to include one where the nOMV az ·, z ^:.

summer

B E2017 / 5855 activated by oxidation of a group one

In another embodiment of the invention, the reaction step is a reductive amination.

As discussed above in detail, the nOMV conjugates of

The invent ion include a fraction s a c ch a r i d i gu of .surface of the nOMV connected directly to a selected antigen

In an equally preferred embodiment, the surface saccharide fraction of activated nOMV is onnected to the selected antigen indirectly, for example, through a will generally be a bifunctional linker, using a functional group to

Intermediary of the activated saccharide fraction) and another functional group to react with the selected antigen * The linker can be a hetero-erobifunctional linker or a homobifunctional linker of general formula (I)

X-L-X '

B E2017 / 5855 in which the groups X and X 'are independently identical, surface saccharide fraction of the activated nOMV and i respectively and

L represents a spacer preferably f o mu l e generated at 1 e (1I.) (II)

TO inde p endammenfc identical or different from each other and feels selected carbonyl group (C ~ O), ester

1o r if i1 c includes at least 3 of: a (- C (O) O ~) or friend of a linear group, (or, a branched cyclic alkyl group. In Cl to CIO having 1 to 10 carbon atoms

C6, 07, C8, 09, C10);

and

L2 represents a linear or branched C1 to C10 alkyl group containing 10 carbon atoms, preferably comprising 4 carbon atoms, even more preferably in the form of a linear chain.

• Ä, a sulfo residue ~ N ~ hydroxysuccinimide and

N-oxys.ucc: optionally substituted inirttide.

both the nOMV and f one t i onne1s, they involve the same groups of

Use a linker where the two identical X groups.

B E2017 / 5855

When the functional groups on the saccharide fraction of nOMV and on the selected antigen are both aldehydes, it is preferred to use a functional linker having X chosen from: a reactive group -NH2, “NH-NH2 or -O-NHj. In an embodiment, still more preferred, the linker is the dihydrazide of adipic acid (ADH) of general formula:

The linker can then be reacted with ££ nOMV and / or the antigen by reductive amination as presented above.

The preferred bifunctional linkers which are particularly useful for the reaction with amine groups of the selected antigen are chosen from: halides, acryloyl, preferably chloride, disuccinimidyl glutarate, disuccinimidyl suberate and ethylene glycol bis [succinimidyl succinate].

Still other preferred molecules are chosen from: the β-propionamido group, neutrophenylethylamine, haloacyl halides, glycosidic derivative bonds, 6-aminocaproic acid.

In yet another: preferred embodiment, the linker is chosen from: N-hydroxysuccinimide, N ~ oxysuccinimide, even more preferably from 3Q from the N-hydroxysuccinimide diester of adipic acid (SIDEA).

Yes

When the reaction with the nOMV and the antigen involves different functional groups (such as an amine on the noMV and a thiol on the antigen), it should be understood that a heterobifunctional linker capable of reacting selectively with will be used. the two different functional groups. In this case, the preferred heterobifunctional linkers are chosen from au. minus one of: succinimidyl 3- ('2-pyridyldithio) propionate (SPDP), succinimidyl hexanoate (LC ~ SPDP) 6- (3- (210 pyridyldithio] -propionamido), 6- (3 '- (2 ~ pyridyldithio) propionamido) sulfosuccinimidyl hexanoate (sulfo-LC-SPDP), 4succinimidylozycarbonyl-α-methyl-a- (2-pyridyldithio) toluene, 6- [a-methÿl.-a- (2-pyridyldithio) toluenamido] solfosuccinimidyl hexanoate (sulfô-LCSMPT), 4- (N-maleimidomethyl) cyclohexane-1 succinimidylcarboxylate (SMCC), 4- (N-maleimidomê thy 1) cyc 1 o hex an e - .1 - carboxy 1 ate de su 1 fosu cc inimi dy 1 e (sulfo-SMCC), the ester of m-raaleimidobenzoyl-N “hydroxysuccinimide (MBS), the ester of m-maleimidobenzoyl-N-hydwxysulfosuGciniraide (sulfc -MBS), the (4iodoacêtyl) aminobenzoate N-succinimidyl (SIAB), the (4-Etyl -iodoac) aminobenzoate sulf osuccinimidyle (sulfo-SIAB), 4- (N-maleimidophenyl) butyrate succinimidyl ²⁵ (SMPB) , sulfosuccinimidyl 4- (N-maleimidophenyl) butyrate (s ulfo-SMPB), the Ν-γmaleimidobutyryl-oxysuceinimide ester (GMBS), 1. Ν-γmaleimidobutyryl-oxysulfosucciniihide ester (sul: fö ~ GMBS), 6- (((((4- (iodoacetyl) amino) methyl) cvc.lohexane-130 carbonyl) amino) succinimidyl hexanoate (SLACK) _f le 6 [6- ((((iodoacety1) amino) hexanoy1j amino] hexanoate

B E2017 / 5855

B E2017 / 5855 succinimidyle (SIAXX), 4 - (((iodoacétyi) amino) ~ methyl) cyclohexane- · 1-succinimidyl carboxylate (SIAC) t and 6 - [(iodoacetyl) amino] succinimidyle hexanoate (SIAX) and 1 ¹ p-nitrophenyl iodoacetate (NPIA).

In another embodiment, the method includes the possibility of recycling the selected antigen which has not reacted particularly when it is in the form of polypeptide. In this way, it has been discovered that the unreacted antigen from the conjugation mixture can be conveniently recycled to the conjugation step, thereby improving the overall efficiency of conjugate production of the invention, and obtaining final conjugates always endowed with remarkable immunogenicity (see the present example 6).

As explained above in detail, the present method allows the preparation of the nOMV conjugates of the invention in a simple and practical manner, also requiring fewer steps compared to the prior methods for the preparation of conjugated derivatives similar (for example, starting from dOMV). Thus, the invention also relates to a conjugate of nOMV obtained (or obtainable) by the method of the invention, according to the embodiments described above. In particular, the present method does not necessarily require the costly step of derivatization of a polypeptide antigen, as well as the absence of carrying out an extraction (for example, using a detergent) or denaturing of the bladder of departure. The production and purification of the nOMVs of the invention are in fact less expensive than

B E2017 / 5855 for conventional support proteins that are more robust and homogeneous than the production of dOMVs. The nOMVs used in the invention can be produced at high yields using, for example, two simple steps of tangential flow filtration, and bypassing detergent extraction procedures. Furthermore, the unreacted selected antigen can be recycled from the conjugation mixture for use in the method, improving the efficiency of preparation of the conjugates, as illustrated in Example 6. Furthermore, the present invention provides: an easy way to prepare a polyvalent vaccine, i.e., a vaccine which includes multiple immunogens

(generally from c the different pathogens) in 15 choosing correctly NOMVs and a int i gene cho i s i as described here p read in detail. In fact, thanks

to its versatility, the present method can be practically and effectively applied to nOMVs from different sources (eg, · Salmonella _r Shigella and meningococcal), by being successfully applied to both protein and saccharide antigens.

Eventually there. It should be noted that the present method not only allows the preparation of highly immunogenic conjugates, but they also significantly change the integrity and size distribution of nOMVs. This is particularly appreciated by those skilled in the art, because the absence of nOMV aggregates allows better yield and overall homogeneity and robustness of the present process.

The present invention is also useful for the preparation of functionalized nOMV conjugates of

B E2017 / 5855 various ways, allowing a multivalent presentation of different antigens on the surface of: the chosen vesicle.

Thus, according to a preferred embodiment,

The invention relates to a conjugate comprising a native outer membrane vesicle as presented above / comprising at least one surface saccharide fraction connected to at least one first antigen, wherein said first antigen is connected to a different second second antigen according to the general formula (I) nOMV —Agi - Ag 2 {I)

In this direction, the two selected antigens (indicated here by Agi and A§2) can be coupled together to give an antigen-antigen derivative (AgiAg.2), which can then be connected to the surface saccharide fraction of the NOMVs 1'intermédiaire chosen by a procedure ¹ reductive amination coxœfte 20 has been described above.

Alternatively, the surface saccharide of nOMV can. first be connected to the chosen GLA via a reducing amination procedure, as described above, to give a nOMV-Agi conjugate, and then a second Ag2 is connected to said nOMV- conjugate Acted, to give the conjugate of the general formula (I) above.

In all cases, the preferred native outer membrane vesicles are GMM.A vesicles, more preferably from Neisseria Meh.B. The Agi and Ag2 can be chosen from the preferred antigens

B E2017 / 5855 as described above, being protein or polysaccharide fractions, Preferably, the antigens used for the multi-functionalization as envisaged here are both proteins or both polysaccharides, or even a protein or a saccharide.

In all cases, the connection between the antigens and the saccharide fraction of nQMV can be made directly, or by using suitable activators, or linkers according to the preferred embodiment described herein.

Thus, according to a more preferred embodiment, the invention relates to a conjugate of the general formula (I) above, in which Agi comprises the protein antigen (NANP) s, Ag2 comprises the protein antigen pfs -25, more preferably by having the nOMV particle obtained from S. typhimurium.

In more detail, said conjugate is preferably prepared by a process comprising the following steps:

a) activation of the Pfs25 antigen using EMCS, to give the activated Pfs25 intermediate indicated below;

r ^-

f:

<>

side $ £ $ 25 'dft ZUEHCS

b) the connection of said Pr.s25 intermediary activated with (NANP> 3 to give the derivative Pfs25-EMCS- (NANP) 3

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c) the reaction of the Pfs25 part of such a derivative with the intermediary of the native external membrane vesicle, by the intermediary of a reductive amination reaction according to the embodiments described above to give the conjugate GMMA ~ Pfs25- (NANP) 3

Western blot analysis confirmed the formation of the conjugate where Pfs25 is connected to the surface saccharide fraction of GMMA, and no aggregation is detected.

According to another embodiment, the present invention relates to an immunogenic conjugate comprising native outer membrane vesicle comprising at least one surface of saccharide moiety connected to a first antigen (AgI) by 1 intermedia ^ir: ed proceedings of aminat i one reduct ri ce as described above, and at least one saccharide surface fraction connected to a second different antigen (Ag2) via a reductive amination procedure as described above above to give a conjugate indicated by the general formula (II):

Agi - -nOMV-— Ag2 (II)

B E2017 / 5855

According to a preferred embodiment, in the general formula (II) above, the .nOMV is. a GMMA from MenB, Agi is a meningococcal capsular polysaccharide from serogroup C, and Ag2 is a meningococcal capsular polysaccharide from serogroup C.

The conjugates of formula (II) can advantageously provide selective raulti-functionalization of nOMVs, using a specific functionalization profile using the reducing procedure will include, te chno1ogi e s e1on 1’i n ven t i on.

proposed, that be used f one tionna1i s t t even with more the possibility of

..on so

Those skilled in the art of the versatility of the present invention may be suitable for the muittdes nOMV, antigens to choose from different types, preferably GMMAs, from different types. Apart from antigens, thus modulating the antigen / nOMV ratio according to, for example, chosen or the nOMV.

According to another aspect, the invention combines nOMV-antigen · described above use as a preferred agent for.

from the

The antigen relates, to for a drug, particularly as an immunogen, in one or more of the homogeneous patterns as indicated i.c

1'in ti on Fri relates to 1 ^st use of the present conjugates NOMVs for the manufacture of a composition

According to another immunogen.

aspect, the invention. thus relates to an immunogenic composition, preferably a

B E2017 / 5855 vaccine, comprising a conjugate of the invention and at least one carrier, an excipient or an additional pharmaceutically acceptable adjuvant. Generally, the pharmaceutically acceptable carrier or excipient may be any substance which does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without excessive toxicity. The pharmaceutically acceptable carriers and excipients are those used in the art, and may include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances and the like, may also be present in such vehicles, according to the prior art.

The invention also provides a method for producing an immune response in a vertebrate, preferably a mammal, comprising: administering a conjugate of the invention to the mammal or other 2.0 vertebrate. The invention also provides conjugates of the invention for use in such methods. The immune response is preferably protective and preferably: involves antibodies. The method may produce a callback response. The mammal is preferably one. To be human. The subject in whom the disease is prevented may not be the same as the subject receiving the conjugate of the invention. For example, a conjugate can be administered to a woman (before or during pregnancy) to protect her offspring (here called "maternal immunization"). The conjugates of the invention can also be used for

B E2017 / 5855 immunize other mammals, for example, cattle, sheep and pigs (especially against Salmonella sp.), And other non-mammalian vertebrates including fish and poultry.

The invention provides u t i 1 i sa t i o n compositions

L ^! invention .immune use

The invention of conjugates (for example, as or as a conjugate for a vaccine).

in therapy; immunogens also provide one in a method of producing a response in a vertebrate, preferably a mammal.

also provides the use of a for a conjugate in the manufacture of a medicament for producing an immune response in a vertebrate, preferably a mammal. The uses and methods are particularly useful for preventing / treating various diseases, depending on the antigens and nOMVs within the conjugates as presented above.

The preferred conjugates of the invention can confer an antibody titer in a patient who is greater than 20. criterion for seroprotection for each antigenic component for an acceptable percentage of human subjects. Antigens with an associated antibody titer above which a host is considered to have undergone seroconversion against the antigen are well known, and such titles are published by organizations such as WHO. Preferably more than 8.0% of a statistically significant sample of subjects has seroconversion, more preferably more than 90%, still more preferably more than 93% and most preferably 96 to 100%.

B E2017 / 5855

The immunogenic compositions of the genera nt administered are di r e cterne n t

Direct administration can be parenteral injection (e.g. subcutaneous, intraperitoneal, intra-muscular, intramuscular, or rectal, oral, topical, trans rMI that pulmonary example, in the thigh or can be performed. using d ¹ a time. hypodermic needle),

i. nt x: a na s al e, or top other eyepiece, muGosaie.

preferred, by a needle (for example, a needle can be used n t conventional scalar but i s es es an injection without variant ”A dose:

about 0.5 ml

The invention can also be used to trigger systemic and / or mu co-s immunity. The treatment can be used in a primary immunization schedule and / or in a booster immunization schedule. A primary dose schedule of a dose schedule t e mp s a p p r op er i ed between doses.

Infections affect various areas of the body and therefore the compositions of the invention can be prepared in various forms. For example, compositions may be prepared under the form of injectable ^r, either liquid solutions or, of

B E2017 / 5855 suspensions. Solid forms suitable for solution, or suspension, in liquid vehicles prior to injection can also be prepared. The composition can be prepared for topical administration, for example, in the form of an ointment, a cream or a powder. The composition can be prepared for oral administration, for example, in the form of a tablet or capsule, or in the form of a syrup (optionally flavored). The composition can be prepared for pulmonary administration, for example, in the form of an inhaler, using a fine powder or a spray. The composition can be prepared in the form of a suppository or an ovum. The composition can be prepared for nasal, ear or eye administration, for example, in the form of drops. The compositions suitable for parenteral injection are most preferred. The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, for example, between pH 6 and pH 8, generally around pH 7. The compositions of the invention can be isotonic with respect to humans. The immunogenic compositions. include an immunologically effective amount of a conjugate of the invention, as well as any other specified component, as required. Dosage therapy can be a single dose schedule or a multiple dose schedule (for example, including booster doses). The composition can be administered in conjunction with other immunoregulatory agents.

B E2017 / 5855

Adjuvants which may be optionally used in compositions of the invention include, but are not limited to, insoluble metal salts, oil in water emulsions (e.g., MF'59 or Α3Θ3, two containing squalene), saponins, non-toxic derivatives of LPS (such as monophosphoryl-lipid .A or MPL 3-O-deacylated), immunostimulatory oligonucleotides, detoxified bacterial ADPribosylant toxins, micropafticles, liposomes, imidazoguinolones, or their mixtures. Other substances which act as immune stimulants are disclosed / for example, in Watson, Pediatr, Infect. Dis. J. (2000) 1'9: 331-332. The use of an adjuvant of aluminum hydroxide and / or phosphate d ^! aluminum is particularly preferred. These salts include oxyhydroxides and hydroxyphosphates. The salts can take any suitable form (for example, gel, crystalline, amorphous, etc.).

The conjugates of the invention which include nOMVs from one pathogen and a selected antigen from a second pathogen may be useful as multivalent vaccines. The pathogen pairs that can be combined (one as an antigen, and the other as a nOMV vesicle) include, but are not limited to: N. meningitidis and non-typhoid Salmonella (e.g., Salmonella. Typhimurium or Salmonella enteritidis); P. falciparum and non-typhoid Salmonella; Salmonella typhi and. Salmonella non-typhoid; ETEC and Shigella sp. ; Streptococcus

B E2017 / 5855 group A (GAS) and N. meningitidis; and GAS and non-typhoid Salmonella.

The preferred pairings of the invention. are shown in Table A below.

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Table A

Preferred nOMV-antigen combinations

NOMVs Antigen f SaImone11a t yphimurium Neisseria meningitidis fHbp Salmone1la typhimurium CSP of Plasmodium falciparum Sa1monalla typhimurium Pf.S'25 of Plasmodium falciparum Salmonel 1 a t yphimurium RO6C of Plasmodium falciparum Sa 1monalla typhimurium ROI OC of Plasmodium: falciparum Salmonella typhimurium CTF1232 of Escherichia coli Sa lm on e 11 a t y p h i m u r 1 um Saccharide Vi from S. t yph imurium Neisseria meningitidis fiibp of Neisseria meningi tidis Ne i s s ri ri m m e n in g i t i d i s Qligosaecharide polyrhamnose Shigella, preferably sonnei CTFI232 d ¹ Escherichia coli Neisseria meningitidis B Capsular saccharide from Men a Neisseria meningitidis. B Capsular saccharide from Men c

Thus, the present nOMV-antigen conjugates are particularly useful as immunogens against the pathogens listed in Table A.

The conjugates of. the invention which. include nOMVs from a pathogen and a selected antigen

B E2017 / 5855 from a second pathogen may be useful as immunogenic compounds for the preparation of multivalent vaccines. Thus, the invention provides a composition comprising a conjugate of: the invention and one or more of the following other antigens:

- a saccharide antigen from

N. meningitidis from serogroup A, C, W135 and / or Y,

- a saccharide antigen from

Streptococcus pneumonia,

- an antigen from the hepatitis A virus, such as an inactivated virus,

- an antigen from the hepatitis B virus, such as surface and / or core antigens,

a diphtheria antigen, such as diphtheria toxoid, for example, the mutant ÇRM197,

- a tetanus antigen, such as tetanus toxoid, ~ an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and

Filamentous hemagglutinin (FHÄ) from

B. pertussis, possibly also in combination with pertactin and / or agglutinogens 2 and 3,

- a saccharide antigen from Haemophilus influenzae Ά or B,

25. ~ one or more polio antigens such as IPV,

- measles, mumps and / or rubella antigens,

- one or more influenza antigens, such as hemagglutinin surface proteins and / or

3Q ne uramin ida se,

- an antigen from Moraxella catarrhalis,

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- a protein antigen from

Streptococcus agalactiae (group B streptococcus),

- a saccharide antigen from

Streptococcus agalactiae (group B streptococcus),

- an antigen from Streptococcus pyogenes (group A streptococcus),

- an antigen from Staphylococcus: aureus.

The invention will now be described by the following experimental part, without placing any limitation on its scope.

Experimental part

Example 1 - · Production of s nOMV

The preferred nOMVs used in this experimental section are GMMAs prepared from AtolR strains of S. typhimuriuia or S, sonnei, for example, as disclosed in Clin. Immunol vaccine. April 1016; 23 (4): 304314 and PLoS One. 2015 ; 10 (8):

60134478 respectively.

The characteristics of said nOMVs were as ¹ indicated in _: Table 2 below.

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Table 2

Characteristics of purified nOMVs prepared from Δ-tolR strains of S. typhimurium or S. sonnei

(ûtplR ûhti-B) Diameter / nfe} 131., 5 14S Surface charge (mV) -14.1 -9.87 Lipid A / ^? vesicle ttig 172.8 155.4 Weighted report OAg / total proteins : Q, BA; Ö:,.: Ö3S:

Example 2 _: - Oxidation of nOMV

Various oxidation conditions have been tested.

For example, for the oxidation of nOMV AtolR 1418, NalCq concentrations in the range of 5 to 20 mM were tested. The effect of oxidation on the chain length of 1'-OAg has been estimated. The length of the chain was reduced with the progression of oxidation.

Higher concentrations of NalO ^ tended to reduce the size of the OAg. It has been verified: that the increase in molarity of NaiO-s has not only led to higher oxidation rates, from 5 to 45% (with rhamnose, the main sugar involved in the process), but also has reduces the length of the OAg chain. At the same time, there was no change in the size distribution of the vesicles, and the integrity of the vesicles was maintained.

This was verified by the dynamic diffusion _of: light (DLS), the analysis of individual monitoring of nanoparticles (NT.A) and liquid chromatography 30 High performance size-exclusion / diffusion of light in multiple angles (HPLC -SEC / MALS; see

B E2017 / 5855 table 2a). It was also: verified that the NHz groups on the nOMVs did not react with the CHO groups produced under amination, reducing conditions producing larger crosslinked particles (Table 2b).

Table 2a

Analysis of oxidized nOMVs (obtained by reaction with 10 and 20 mM NalOi) by DLS and HPLC-SEC / MALS

Sample (z-moyon) / (Pdl) Ί (nm by: L.LS): l ·  (nm par. EVIL 3) nOMV 1418 AtolR 131.5 (0.219) 1 30.5 nOMVox 141.8 AtolR 129.5 (0.273) 27.4 3.0 mM nOMVox 1418 AtolR 20 mM 135.8 (0.224) ί 30.5

Table 2b

No reaction of nOMV under reducing amination conditions, verified by DLS and HPLCSEC / MALS

p Sample Diameter · (z “hub) (Pdl) nm pale · DLS ^: (nm pa r MALS · :): /:. 1 nOMVox 1418 AtolR 1 20 mM NaIO ₄ 135.8 (0.224) 30.5 1 nOMVox 1418 AtolR j 20 mM NaIGu after i reaction with 1 NaBHaCN 131.7 (0.291) 31.2

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Under reductive amination conditions, without nOMV, the foreign antigen to be conjugated gives no aggregation, verified for several antigens (for example, fHbp, Pfs25 and CTF1232).

In. Furthermore, it has been verified, that NaBRsCN does not reduce SS bonds, which could otherwise affect the conformation of proteins such as Pfs25, Pfs25 was treated with NaSHaCN under conditions mimicking conjugation, and the same reaction was carried out with DTT as a reducing agent for comparison. After mixing overnight at room temperature, analysis by SDS-PAGE and HPLC-SEC of the Pfs2 5 treated with NaBHs 2, in contrast to the Pfs 25 treated with DTT, did not show any change in the protein comparatively. with fresh Pfs25. The same results were confirmed by MAL.DIMS analysis, when the protein was treated with 1 i iodoa-ketamide (IAÄ) in the presence of NABfhCN or DTT.

20- Other experiments on nOMV of S. typhimurium triple mutant (nOMV of Sh typhimurium 2192 ÛtolR ûPagP AmsbB) showed that low concentrations of NalCM (3 to 5 mM) were also sufficient to obtain good oxidation rates . Examples of results are presented in the. Table 2c, using a concentration of nOMV in the range of 0.2 to 4 mg / ml, a pH in the range of 5 to 8, and a concentration of NalQ "in the range of 0.5 to 5 mM. The resulting vesicles were estimated for% recovery of nOMV,% oxidation of rhamnose, size of nOMV, and la. size of the QAg.

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Table 2c

Oxidation of riOMV of S. tyiahimuriiim triple mutant

j Sr..ie iilll Postman ////// /////// 1: / /// :? άί: ίί $ ^ ΐ / <2 / ί / Postman ////// ///// i :: // RépOÎJSe ^: Answer: ///////////// 2 // ::: /: /: /: /: / ::: Reply Répons # ^ 17777 ^ 77777 ^ Ά: inOMV] to rîWXW etpH * laughs iMicro BOA nOMV recovery) â * oxidation from Rha : iWÄFC « ADF) Cut of. NOMVs (r medium by ÖLS) Cut: from 1 : (M0 4RIÏ pg / IUL tril · · I1 1 ΠίϊΙ koa 2100 2.7 5 6.5 72 io 50,28- 20907 II / ssp / s / isi : ^:: 21W 2.75 B 75 6 50.77 24215 ï / ss / d / s / SI • looe 0.5 5 7§ 0 51.52 31148 suii " nw 2/75 5 80 13 48.09 17935 : /////// s / 5 ///////// 200 5 5 8 7: 58. 42.39 682 b '' R / '/ 2100 2.75 5/5 77 '7 50: ^4:: 20276 ghyl / s ^ çw Λ> 5 6, 5 ': .8: 1- 3 50, 89 25221 If® " : 2100 § 6.5. 82 2.3 47.56 10170 : 1O: : 2: 1.0: 0 2.75 v _f 5 80 10 4 9, 8 184 93 ïsssllssss 2® " 0.5 / es s 85 4 50, 3 9 3: 0392 ///////; i _: 7/7 ///// 2.00 0.5 5 83 : 7 ' 50: 1: 9: 25131 isissia / ii / i 2100 2 75 ^: h, 5 82 9 N / A, 85 18: 493: /"ie 2100 2.75 6/5 78 lô: 49.07 20589 "" "" 20O 0.5 8 77 51.42 _:: 30'347 yae 200 vs. • 8: 72 2OS 47, es 1.0980 //// 517 /// '// 2100 2, 75 b, 5 83 B 49/09 20536

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j ^J al ...... 4ÖÖÖ! 5 & 80 11 1 4 8 _f 4 4 3 8212 4000 -0: 5. '8 ; JL j 50, .8-6 · 30208 § M il · //////// · i .................. _.. ................ i 1 4000 d ⁵ 86 is wen

5:

Similar nOMV recovery rates were observed for all reaction conditions, tested by microassay of proteins with bicinchoninic acid (micro BCA). It was further verified θ that none of the reaction conditions tested gave rise to crosslinking or aggregation of nOMVs.

The percentage of oxidation of the rhamndse was affected by both. nOMV concentration and _r NaTCg concentration, with a lower concentration of nOMV and a higher concentration of NalO "tending to give higher levels

oxidation of rhamnose. By con: segregate, in general, the nOMV concentration and / or concentration of NalCM can be manipulated to obtain : set the desired rate of the date of rhamnose (or of a other sugar). To compare vesicles with a size of-OAg and rhi oxidation rates ïmnose- different, the vesicles of series 5, 14 and 31 6 of Table 5 were

treated with NaBH "to remove the aldehyde groups (CHO) and stabilize the nOMV vesicles, and the rate of rhamnose oxidation and the size of the OAg were again estimated after treatment. The results are presented below in Table 2d.

Table 2d

Treatment with NaBEU series 5, .14 and 16

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Conditions for „1 'ôxydati'J _: tr S: d ¹ oxidation of Ria expected Size da 1 'OAg expected Pre-reduction P-ö st. “Ré du and i en %: d ¹ oxidation of Rha expected Size of i> oag expected e % of Se Kha oxidation: expected Size of 1 ¹ oag expected e 5 series 58 6826 Da 58 7481 of 53 6031 Da Series 16 : rs 30 ^ 80 p at 14.4 1STS? 0 16 16528: D at Series 14 10 20589 » at 13 24224 D at 12 21163 P AT

The results show that the reduction step did not affect the length of 1 ’-OAg or the degree of oxidation of the nOMVs. Similar results were obtained in a separate experiment.

Example 3 - Conjugation step nOMV-Ag ______ (fVi-nOMV ..... de

S. typhimurium; conjugation_____indirect by

1 through a linker)

The fVi was modified by reaction with an ADH linker, either randomly (r) or selectively (s). The modified fVi was then conjugated: to nOMVs of S. typhimurium oxidized using a reductive amination. When the fVi was activated randomly with .1 'A DH, a weight ratio fVi: on nOMV of .1 / 1 was used in the conjugation, while a weight ratio of 3/1 was used when the fVi has been derivatized at its end with DHA. The conjugates were characterized using the micro BCA / Lowry test to determine the. total protein content (recovery of nOMV); high performance anion exchange chromatography with pulsed amperforetry detection (HPAÉC-PAD) was used to determine the total Vi content (no interference

B E2017 / 5855 nOMV); high performance liquid chromatography-exclusion chromatography (HPLC-SEC using a gel column TSK 3000 PWxl) was used to estimate the% of free Vi using the differential refractive index (dRI) and the diffusion light dynamics-chromatography, high performance liquid-exclusion chromatography static light scattering from multiple angles (DLS / HPLÇ-SEC-MALS) was used to determine the size. The effect of the conjugation conditions of this conjugation process on the final number of fVi chains by nOMV was estimated by considering the data collected by HPAEC-PAD as well as the number of nOMVs by the analysis of individual monitoring of nanoparticles. (NTA).

The results are given in Table 3.

Table 3

Influence of NaIO <concentration and pH on cen j. a gu 6s f Vi-nOMV

Series: FLW " r / s î de .c; Lv / l / [S-I: /// OIJ: : pH: ld: ///////:%/:of///:::/:::/:: /: Rdppöit: / Isft'Säijiis :: /: / 11611311 average from iVi 11ι11ΐι £ θΤ / ^: / ι / ί / ί con jugsi.s on récupératidn (I / i / p / p / :::: /: // idé / TVi / :::::: GCIU / pIf: / :: / syëtsvéëS /: ^: . Οχ y dà-ii ca FxCW ivi / pp, t ^; e • IL / itsel (r) /% /// PEA / AOO / :: // / (/ FT® :: ::::::: '1: 3110/1111/11: ilt / l / i / i //// 77777/771: ////////// <ie / // < NOMVs chains VV / <ifôiiijVÎ //// activated ////// ILJ /: ///// ' 7 (1/17 /// //// 48.5 'd> · 24.4 20: : 4: 5. 30, 7 : 07'4 5:: 97 48, fi t 24.4 see you 6 43, 8 0.35 /7.6/ /: The //// //// 48.5 r 30, Ί 20 7.2 58, S 0.2 : 43--: /: /// ": //: /// 48.5 / g > 95 10 -7.2 69 Ö: _f : S8 17 : / :: / 5II ::: ::: /: /: 48.5 > 95 30- 4.5 48, 9 Ö, 44 95 / 7 ^ /// 11 // 17 48.5 S >: 35: 10 4.5 80 0.43 93 / ::::: / 7: / ::: / ::: /: 23! r .11 20 4/0 80 0.08 3: 7

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23 X there : 1 " 4, 5 89.4 _: 0.07 32 : Ss: 9i / sss 23 there 11 10 ' t 37 <0.04 18 ß r 23.8 1Ö 4 <5 1001 _: 0.12 153 VPCP 3, 8 r 15.5 20 4, 5 100 .0,05 138 3.8 r 15, -Ÿ : 1Ö "LS · ............ 67, 6 0 / 0'7 Cl 94: PBC 3, 8 r 15.5 10 .6 82 0.02 ace i. 3.8 r 16.2 10 3.2 / i 74 _: 0,: 01 30·

r: random introduction from the inside (Aßii) along the fVI * antigen.

s: selective introduction of the linker (ÄDH) at the terminal end of the fvi antigen.

RO: repeating units.

W Average uesLipe pi '4! IΓ a fits and 3, years -n 4 't' an ”.

Carrying out the reductive amination at pH 4.5 with fVi at 48.5 kDa, produced higher ratios of fVi / nOMV and more chains of fVi per nOMV at both 10 mM and 2.0 mM ICI, compared to a higher pH ·. With randomly modified fVI, precipitation occurred at pH 4.5; precipitation was avoided by working with selectively modified fVi. In general, selective chemistry has been identified as a means to improve recovery of nOMVs and to avoid precipitation. No precipitation was observed for fVi with an average MM <2'3 kDa at. low pH with fVi chains changes randomly ...

Oxidation of nOMVs with 10 mM NaIO, ₃ instead of 20 mM resulted in improved recovery of the conjugates. The increase in NaTOa concentration had no impact on the characteristics of the final conjugates.

When using it. from fVi to 23 kDa, lower ratios of fVi to nOMV have. obtained, which may have been associated with the lower derivative percentage of fVi-ADH used for conjugation (11% of repetitive units of fVij ...

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The pH and the degree of activation of fVi-ÀDH have been identified as variables for the modulation of the number of fVi chains linked by nOMV,

Example 3a - Vi-nQMV conjugates of S. typhimuriiim (indirect conjugation via a linker) Conjugates 1 and 5 according to Table 3 above, having Vi / nOMV p / p ratios of 0.45 and 0.44 respectively were tested in mice. For comparison, CRM197 was also used as a carrier, and a simple mixture of fVi + nOMV was also tested.

Mice were immunized subcutaneously on days 0 and 28 with the conjugates (dose of 1 μg of 15 Vi) and an adjuvant Alhydrogel. AntiVi IgG titers were measured on days 0, 14, 28 and 42, and the results are shown in Figure 2A. The nOMV was not lower than the CRM197, but it was significantly better than the unconjugated mixture.

IgG titers against 1'-OAg were also estimated. FIG. 2B represents the anti-OAg titers for the vesicle alone or conjugated to Vi. Conjugation reduced the response to OAg by a small amount, but the responses remained significant.

Example 4. - Conjugation step of nOMV-Ag (CTF1232 from ETECnOMV ..... from S. typhimuriurn AtolR 1418 or S. sonné!

1790; direct conjugation without linker)

CTF1232 is an E antigen. collar! (SEQ ID NO: 14

3.0 with a C-teriainal marker for hexa-histidine which has been conjugated to the two types of nOMV vesicles. The polypeptide

B E2017 / 5855 contains five residues of .lysine which can be used for binding to oxidized saccharides in vesicles.

For conjugation to CTF1232, the vesicles were oxidized in 100 mM sodium acetate (pH. 5) with

sodium periodate (20 mM for S.

typhimuri um _f 40 ràM for S. sonnei) for 2 hours in the dark at room temperature. Oxidation in S. typhimurium a. been preferential in the. ihamnose residues (Rha), with, approximately 30% of oxidized Rha units (calculated by

1Q compared to mannose). Oxidation in S. sonnei had an impact on the nucleus region of _: LPS molecules.

500 pg of oxidized vesicles (measured proteins) from nOMV or from S. typhimurium ûtolR

141.8 or S. sonnei 1790 were reacted with

500 pg of CTF12 32 with 1 to 2 mg of NaBlMCN at room temperature over a weekend. Based on the quantification of the antigen which did not react after conjugation, Here presence of the antigen on the surface of the nOMV was calculated as being <36% âaiis

S typhimurium and <31% in S. sonnei.

The results showed that the chemistry of the reductive amination is suitable for the conjugation of polypeptide antigens to the vesicles. The CTF1232 antigen was conjugated to the oxidized LPS of the two vesicles.

Mice were immunized with the protein alone,

a mix of. protein ne and nOMV of Shigella _r or the conjugates. The adjùvar it Alhydrogel was used in all groups. Will read them ammunition were administered even intranasal route e two days 0, 21 e- t 38 and the

immune responses were estimated on days 0,: 14,

B E2017 / 5855 and 52. The anti-CTFl232 IgG titers are presented in FIG. 3.

On day 14, the nOMV 1418-CTF1232 conjugate was able to induce a significantly superior response to the protein alone (p ~ 0.0.005) or physically mixed with nOMV (p - 0.042) (KruskalWallis test with post analysis -hoc de Dunn). In addition to improving anti-CTF1232 titers, the conjugate had the additional benefit of being a bivalent XQ vaccine. No major differences were observed between the nOMV 1790-CTF1232 and nOMV 1418CTF12 32 conjugates, meaning that the nOMVs of both Salmonella and Shigella can function as good carriers for the ETEC antigen.

Example 5 - Conjugation step of nOMV-Ag (conjugation of P f s 2 5 -nOMV of S. typhimurium in the presence of NagSOa),

The malaria antigen Pfs25 has been conjugated to the nOMV vesicles of S. typhimurium by two different chemistries: to proteins via SH-maleirnido or chiral click ƒ groups or to OAg oxidized by 'NalOi, · For the binding to oxidized 1'OAg, a 1/1 ratio of nOMV / Pfs25 was used, at a Pfs25 concentration of 2.6 mg / ml in PBS with overnight incubation at room temperature. Conjugate formation was also obtained when excess NaI0 ₄ was neutralized with. NaaSCb (a concentration of 10 mM was used in this experiment, for 10 minutes), this followed by the direct addition of Pfs2.5 30 in the same container (final concentration of 0.2 mg / ml). FIG. 4A represents the IgG titers

B E2017 / 5855 ànt-i-Pfs.25 éii reply to: the three conjugates; Pfs.25 alone; or Pfs25 physically mixed with the vesicles. All constructions were formulated with Alhydrogel. The mice were immunized subcutaneously on days 0 and 28 at 0.1 µg of

Pfs25 / dose. The anti-Pfs25 IgG titers were measured on days 0, 14, 28 and 42,

Pfs25 alone induced an antibody response

Anti-Pfs25 IgG significantly lower than the 10 nOMV-SH-Pfs25 conjugates (p ~ 0.001) and nOMV-ox-Pfs25 (p = 0.00 95). Pfs25 physically mixed with nOMV similarly induced a lower response compared to nOMV-SH-Pfs25 and nOMV-ox ~ Pfs25 (p ~ 0.0038 and p ^:::: 0.0282 respectively J (Kruskal15 test Wallis with post-hoc analysis of

Dunn). In a way

Here Pfs25 linked by

The intermediary of the sugar component on nOMV (nOMV-ox-Pfs25 conjugate) induced an antibody response similar to Pfs25 bound to proteins on nOMV (nOMV-SH-Pfs25 and nOMV20 ciick-Pfs25) and higher than the Pfs25 alone or physically mixed with nOMV.

Sera from Pfs25-nOMV conjugates showed transmission blocking activity when analyzed by the SFMA test (standard 25 membrane-feeding assay; transmission reduction activity> 90% at 1: 1 dilution). 8 and maintained at a 1/16 dilution for nQMV-SH-Pfs25 and nOMVox-Pfs2 5).

Binding to Pfs25 on nOMVs had no impact on the anti-OAg IgG response. In addition, the conjugate obtained by reductive amination, where the chemistry

B E2017 / 5855 used has an impact on the structure and the length of the -OAg, maintained high titers of anti-OAg IgG "Consequently, the presence of a foreign antigen on the nOMV of S. typhiraurium. has no impact on anti-OAg IgG responses (see Figure 4C).

In a second study, the immunogenicity of the Pfs25-nOMV conjugate (produced by reductive amination) was compared with Pts2.b physically mixed with nOMV at a dose of 1 μg of Pfs25 without Alhydrogel.

FIG. 4B represents the antiPfs25 IgG response induced in mice by the Pfs25-nOMV conjugate compared to Pfs25 physically mixed with nOMVs without Alhydrogel, using the same immunization schedule as for FIG. 4A.

The conjugate was able to elicit a response by

Anti-Pfs25 IgG significantly superior to the protein mixed with the nOMVs (p = 0.0002; bilateral Mann-Whitney analysis).

Example 6 - Conjugates RO6C-nOMV of S. typhimurium (recycling step)

The Plasmodium R6 antigen was conjugated to oxidized nOMV vesicles of S. typhimurium using reducing amination. Another conjugate was _: produced by recycling the unreacted RO6C from the first conjugation batch and reusing it for conjugation. The ratio of RO6C to total protein was measured by a competitive ELISA test, and it was 7.2% for the non-recycled conjugate and 11.1% for the recycled conjugate. For comparison, the RO6C alone

B E2017 / 5855

at. been used. All; constructs were formulated with Alhydrogel.

Mice were immunized subcutaneously on days 0 and 28, and doses of 1, 4 and 20 µg of RO6C 5 were used. The recycled conjugate was tested at a dose of .4 pg of RO6C. Anti-R06C IgG titers were measured on days 0, 14, 28 and 42, and the results are shown in Figure SA. On day 42, a higher anti-R06'C IgG response was induced by the nOMV-RO6C conjugate compared to RO6C alone (MannWhitney test, p · - 0.05 at a dose of 1 pg, p = 0, 03 at a dose of 4 pg and p - 0.04 at a dose of 20 pg). In addition, the nOMV-RO6C conjugate elicited an antiRQ6C IgG response in a dose-dependent manner (Spearman rank, 15 p === 0.001, day 42). All constructs (at all doses) showed the ability to stimulate the response (day 14 to day 42). The non-recycled and recycled conjugates at a dose of 4 μg were compared by the bilateral Mann-Whitney test, showing the capacity of the recycled conjugate to induce a response which is not less than the non-recycled.

The titers of IgG directed against OAg were also estimated. FIG. SB represents the titers of anti-OAg IgG for non-recycled and recycled vesicles. The doses of the nOMVs corresponding to the doses of RO6C of 1, 4 and 20 pg were 13 pg, 52 pg and 258 pg, respectively. For the recycled conjugate, the dose of nOMV was 32 pg (corresponding to a dose of R06C of 4 pg).

Examples la to c - Comparative examples

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Example 7 a - Reaction of dOMV (from Neisseria meningit.id.is B) with v3. fHbp (no reaction)

The dOMVs of the present example were prepared by a detergent extraction process, where the deoxycholate is used as the selected detergent. The vesicles extracted with a detergent-thus obtained were reacted with the selected antigen (fHbp) according to the method of the present invention. In particular, 10 dOMVs, at a concentration of 0.96 mg / ml, were incubated with NaIO ₄ at 10 mM for 30 minutes: at room temperature, in the dark. The excess NalO / .. was neutralized with NaaSO3 at a final concentration: of 20 mM, for „15 minutes at room temperature. The fHbp (w / w ratio of dOMV to fHbp of 1/1 and with a dOMV concentration of 0.335 mg / ml) and NaBlbCN (3 mg) were added directly to the reaction mixture. After gentle mixing overnight at room temperature, the conjugate was purified by ultracentrifugation (110,000 rpm at 4 ° C for 1 h), resuspended in RBS and analyzed: by SDS P AGE / We sternb 1 Ot

Example 7b - Reaction of nOMVs (from Àtefsserfa 25 meningitidis B) with fHbp v3 (formation of the nOMV ~ fHbp conjugate of the invention) The nOMVs of the present example were prepared without using any detergent, as described in Koeberling et al. Vaccine (2014): 32: 2688. The

3.0 extracted vesicles thus obtained were reacted with the selected antigen (fHbp) according to the method of

B E2017 / 5855 presented invention. In particular ^. nOMVs at a concentration of 0.96 mg / ml were incubated with 5 mM NalOi for 30 minutes at room temperature, in the dark. The excess NaTCM was neutralized with NaPSOa to a final concentration of 20 mM, for minutes at room temperature. The fHbp (w / w ratio of the dOMVs on the fHbp of 1/1 and with a dOMV concentration of 0.335 mg / ml) and the NaBH 3 CN (3 mg) were added directly to the reaction mixture. After gentle mixing overnight at room temperature, the conjugate was purified by ultracentrifugation (110,000 rpm at 4 ^: C for 1 h), resuspended in PBS and analyzed by SES PAGE / We s tern blet . SDS PAGE / Western blot: anti'-fHbp analysis confirmed conjugate formation by. reducing amination only with dOMVs, but not with dOMVs. SDS page freeze at 10%.

Example 7c Reaction of nOMVs (from Salmonella)

20 with vl from fHbp, in following the procedure of Example 7b The same experiment as example 7b was carried out using nOMVs originating from Salmonella typhiwurium, and similar results were collected, obtaining the nOMV-fHbp conjugate of the invention. Example 8 - Preparation. of IONIZED MULTI-WHIP NOMV

using the (NANP) 3 ~ SH-Pfs25, according to the invention

The Pfs2.5 protein has been derivatized with the linker

EMCS according to the following procedure. Pfs25, in

B E2017 / 5855 PBS buffer at a concentration of 2.6 mg / ml, was added with the EMCS linker (molar ratio of the EMCS linker on the Lys residues of Pfs25: 0.3). The reaction was mixed at room temperature for 4 h. The resulting derivatized protein (Pfs25 ~ EMCS) was purified by PD10 column against NaHsPCM at 10 mM pH 6.

Analysis by MALDT-TOF MS revealed an average of 4 EMCS linkers introduced per molecule of Pfs25. The (NANP) s was added to the solution of Pfs25-EMCS for XQ to have a molar ratio of (NANPja on the EMCS linkers of 3/1 and a concentration of Pfs25 of 0.7ng / ml. The reaction was mixed with room temperature overnight. After this time, the Pfs25- (ΝΆΝΡ) 3 derivative was purified by Vivaspin 10K against buffer PBS. Analysis by SDS PAGE / Western blot and MALDI-TOF MS confirmed the formation of nOMVs of S. typhimuriuin at a concentration of 2.1 mg / ml in 100 mM MaHaPO 4 pH 6.5 were incubated with 5 mM Nal'Oa for minutes at 2.5 ° C. in The excess of N.aIO <2Q was neutralized with NasSOs at a final concentration of 10 mM, for 10 minutes at room temperature, the

Pfs25- (NANP) 3 (w / w ratio of nOMVs on Pfs25- (NANP) 3 of 1/1 and with a nOMV concentration of 0.45 mg / ml) and NaBHjCN were added directly to the mixture:

reaction. After mixing, delicate overnight at room temperature, the conjugate was purified by ultracentrifugation (110,000 rpm at 4 ° C. for min), resuspended in PBS and analyzed by SDS PAGE / Western blot, mistletoe. confirmed the formation of 3q: conjugate.

B E2017 / 5855

Example 9 In vivo data of the conjugates of the invention

obtained by conjugation of a particle NOMVs of S. t yphimuri um at the antigen do Pfs25 with or without neutra1isation, according to embodiments of 1'invention Mouses CD1 females have been immunized through way

subcutaneously on days 0 and 28 with 2.5 μg of total proteins of the nOMV particles of S. typhimurium conjugated to the Pfs25 antigen with or without the neutralization step (see example 5}. The two conjugates showed a w / w ratio of Pfs25 on total proteins close to% by a competitive ELISA test and they were adsorbed on Alhydrogel (0.7 mg / ml of AI ³⁴ ). The anti-Pfs5 IgG titers and anti-OAg were measured at 15 days 0, 14, 27 and 42. At all time points, the two conjugates induced a similar response in anti-Pfs25 IgG (Mann Whitney test), as shown in Figure 6a In addition, the conjugates were able to induce a similar IgA anti ™ OAg response, as shown in Figure 30b The neutralization step in reductive amination conjugation can be introduced without any impact on the immune response induced in mice, thus avoiding the purification of i 25 GMMA oxidation intermediates.

Example 10 - Reaction of ....... nOMV ______ (from ....... of ...... Nei s seriala meningitidis B) with MenC (formation of the nOMVMenC conjugate of the invention)

3q: The MenC polysaccharide was dissolved in 100 mM AcONa pH 4.5 at a concentration of 40 mg / ml.

B E2017 / 5855

The ADH linker and NaBHaCN were added in a w / w ratio of 1 / 1.2 / 1.2 of MenC / ADH / NaBHaCN respectively, The mixture was heated at 30 ° C overnight, and then desalted by a G1O column. Characterization 5 by the TNBS colorimetric method and HPAEC-t'AD showed 100% derivatization.

The MenB GMMAs overexpressing Here fHbp, at a concentration of 8.5 mg / ml in NaHaPCq at 100 mM pH 6, were incubated with KlälO “at 5 mM for 3.0 minutes at room temperature, in the dark. The excess NalCy was neutralized with Naj.SOs at a final concentration of 10 mM, for 15 minutes at room temperature. The MenC oligosaccharide, previously derivatized at its end by the ADH linker (ratio, w / w

15. GMMAs on MenC of 1/10 and with a GMMA concentration of 7.7 mg / ml) and NaBHsCN were added directly to the reaction mixture. After gentle mixing overnight at room temperature, the conjugate was purified by ultracentrifugation (110,000 rpm at 4 ° C for 1 h) and resuspended in PBS. Analysis by SDS PAGE / Western blot confirmed the formation of the conjugate and analysis by micro BOA and HPAEC-PAD revealed a weight ratio of MenC polysaccharide on the protein equal to 0.11.

Example 1.1 - Reaction of nOMV _______ (from Ne.isser.ia meningitidis B) with MenA (formation of the nOMVMenA conjugate of the invention)

The MenA bone was solubilized in AcONa at 100 mM pH 6.5 at a concentration of 40 mg / ml. The ADH linker and NaBlhCN were added in a w / w ratio of

B E2017 / 5855

1./.1,2/1,2 of MenA / ADH / NaBHaCN respectively. The mixture was heated at 30 ° C for 5 days, then desalted by a G10 column. Characterization by the Golorimetric method at TNBS and. HPAEC-PAD showed 90% derivatization. Conjugation to the GMMAs of Men.B overexpressing fHbp was carried out as described for the polysaccharide of MenC.

Example _ _ 11 - Preparation of the conjugates of nOMV (is sues 10 of Neisseria meningitidis B) with MenA and MenC

The same conjugation conditions described for the synthesis of the MenA and MenC-MenB-GMMA conjugates were used for the conjugation of the two polysaccharides on the same GMMA particle. The GMMAs were oxidized as described above and, after neutralization with NazS'Oy, the MènA-ADH and MenC-ADH were added simultaneously in a w / w ratio of 8/8/1 Me hA / Me-nC / GMMA- re s pe and i. even.

0

B E2017 / 5855

Sequence list> SEQ ID NO: 1 [v2 of fHbp]

VAADIGAGLADALÏAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKN DKVSRFDFiRQIEVDGQLlTLESGEFQlYkGDEKGKQKKKFKQDQFKGDQFKQDQFKQKQKFQGFQKFQGDQFQGKQKFQGKDQKFQKFQGKQFKQKQKFQGKQKFQFQFKGQKFQGKQQFKQKQQFQFKQKQFKQKQFKQFKQFKQFKQFKQQFQFQFQGFQFQFQGFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQFQQFQQFQFG

DEKSHAViLGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHElGiAGKO> SEQ ID NO: 2 [NHBÄ]

MFKRSVIAMACiFÂLSACGGGGGGSPDVKSADTLSKPAAPWSEKETEAKEOAPQAGSQGQG

APSAQGSQDMâAVSEENTGNGGAVTADNPKNEDEVAQNDMPQNAAGTDSSTPNHTPDPNM "LAGNMENQATOÄGESSQPANQPDMANAADGMQGDDPSAGGONAGNT.AAQGANQAGNNQA ^iV AGSSDP PASNPAPANGGSNFôRVDLÂNG {\ Æ.rDGPSONITLTHCKGDSCSGNNFLDEÉVÔLKS

ÊFEKLSDÂÔKlSNYKKDGKNDKFVGLVADSVQMKGINOYIfFYKPKPTSFARFRRSÀRSRRSLP

AEMPLIPVNQADTLIVDGEAVSLTGHSGNiFAPEGNYRYlTYGAEKLPGGSYALRVQGEPAKGE

MLAGAAVYNGEVLHFHTEHGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIÖG

NGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD> SEQ ID NO: 3 [NadA] mkhfpskvlttailaifcsgalaatsdddvkkaatvaivaaynngqeîngfkagetiydigedgti tqkdataadveaddfkglglkkvvtnltktvnenkqnvdakvkaaeseieklttkladtdaala DTÔAALDETTNALNKLGENlTTFAEETKTNiyKIDËKLEAVADTVDKHAEAFNOlADSLOETNTKA DEAVKTANEAKQTAEEtKQNVDAKVKÂAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIA tnkaûiâknsàridsldknvanlrketrqglaeqaalsglfqpynvgrfnvtâavggyksesa VAÎGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYÊW> SEQ ID NO: 4 [NspA]

MKKALAILIALALPAAALAEGASGFYVQADAAHAKASSSLGSAKGFSPRiSAGYRINDLRFAVDY tryknykapstdfklysigasaiyofdtqspvkpylgarlslnrasvdlggsdsfsqtsîglgvl TGVSYAVTPNVDEDAGYRYNYÎGKVNTVKNVRSGELSAGVRVKF> SEQ ID NO · 5 [NhhÂ] ^ ²⁵ MNKlYRÎfWNSALNAW AAZSELTRNHTKRASATVKTAVLATLLFATVQÂSÀNNEEQEEDLYLGP VQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEkGVLTAREÎTLKAGONLKiKQNGTNFTYS lkkdltdltsvgteklsfsangnkvnitsdtkglnfmetagtngdttvhlngigstltdtlînt GÀTTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDFVRTYÔTVEFLSAOTKT TTVNVESKDNGKKTEVKlGAKTSVIKEKbGkLVTGKDkGENGSSTDEGEGLVTAKEVIDAVNKA GVVRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATVSKDDQGNIîVMYDVNVGDALNV NQLQNSGWNLbSKAVAGSSGKVISGNVSPSKGKMDETVNÎNAGNNfEITRNGKNlDIATSMTPQ fssvslgagadaptlsvdgdalnvgskkonkpvritnvapgvkegdvtnvaqlkgvaonlnn RlDNVDGNARAGIAQA ATAGLVQAYLPGKSMMAIGGGTYRGEAGYAiGYSSISDGGNWHKGT!

ASGNSRGHFGASASVGYQW

B E2017 / 5855> SEQ I'D NO; 6 [App]

MKTTDKRTTETHRKAPKTGRIRFSPAYLAiCLSFGll.PQAWAGHTYFGfNYQYYRDf'AENKGKF

AVGAKDiEVYNKkGELVGKSMTKAPMlDFSWSRNGVÄALVGDQYIVSVAHNGGYNNVDFGÄE

GRNPDQHRFTYKiVKRNNYKAGTKGHPYGGDYHMPRLHKFVTDAEPVEMTSYMDGRKYlDQN

NYPDRVraGAGRQAA'RSDEDEPNNRESSYHIASAYSWLVGGNTFAqNGSGGGTVNLGSEKIK HSPYGFLPTGGSFGDSGSPMFÎYDAQKQKWLINGVLQTGNPYfARKSNFNGNFNQRNDGKRVDFNGKRVDGNRGDGVNFKNVGFNQGNRGDQVNGFNN

AäGGVNSYRPRENNGE'MISFIDEGKGEULTSNINQGAGGLYFQGDFTVSPENNETW.QGÄG.VHI

SEDSTVTWKVNGVANDRLSKIGkGTLHVQAKGENQGS.fSVGDGTV! LDQQADDKGKKQAFSEi

GLySGRGWQLNÄDNQFNPDKLYFGFRGGRLDLNGHSL.SFHRlQNTDEGAMIVNHNQDKEST

VTJTGNKDiATTGNNNSLDSKKElAYNGWFGÉkDTTKTNGRLNLVYQPAAEDRTLLLSGGTNLN

GNiTQTNGKLFFSGRPTPHAYNHLNDHWSOKEGiPRGEIVWDNDWINRTFKAENFQIKGGQAV

VSRNVAKVKGDWHLSNHÂOAVFGVAPHQSHTICTFiSDWTGLTNÇVEKTITDpKVîASLTKTDfô gnvdladhahlnltglatlngnlsängdtrytvshnatqngnlslvgnaqätfnöatlngnt

SASGNASFNLSDHAVQNGSLTLSGNAKANVSHSALNGNVSLADKAVFHFESSRFTGQISGGKD

TALHLKDSEWtLPSGTELGNLNLDNATrrLNSAYRHDAAGAQTGSATDAPRRRSRRSRRSLLS

VTPPTSVÉSRFNTLTVNGKLNGQGTFRFMSËLFGYRSDKLKLAESSEÔTYTLAVNNTGNEPAS

LEQLTWEGKDNKPLSENLNFTLQNEHVDAGAWRYQLIRKDGEFRLHNPVKEQELSDKLGKAE AKKQAEKDNAQSLDALIAAGRDAVEKTESVAEPARQAGGENVGIMQAEEEKKRVQADKDTÄIA KQREAETRPATTAFPRARRARROLPQLQPQPQPQPQRÖLISRYANSGLSEFSAtLNSVFAVQD ELDRVFAEDRRNAVWTSGiRDTKRYRSQDFRAYRQQTDLRQIGMQKNLGSGRVGILFSHNRT

ENTFDDGÏGNSARLAHGAVFGQYGIDRFYIGISAGAGFSSGSLSDGIGGKIRRRVLHYGIOARYR

AGFGGFG1EPHIGATRYFVQKAÔYRYENVNIATPGIJ \ FNRYRAGîKADYSFKPAQHISITPYLSLS yïdaasgkvrtrvntavlaqdfgktrsaewgvnaeikgftlslhaaaakgpqleaqhsagikl GYAW>

ATNDDDVkKAATVÂlAAAYNNGQEïNGFKAGETÎYOlDEDGTITKKDATAADVEADDFKGLGLKK

VVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALAÜTDAAL.DATTNALNKLGENITTF

AEETKTNIVKÎDEKLEAVÀOTVDKHAEAFNDlADSLDETNTkADEAVKTANEAKQTAEETKQNVD ÂKVKAAETAAGKAEAAAGTANTAADKAEAVAAkVTDiKADiATNKDNIÀKkÀNSÀDVYTREESD SKFVRIDGLNATTEKLDTRLASAEKSiAÖHDTRLNGLDkTVSDLRKETRQGlAEQÄALSGLFQP YNVG> SEQ ID NO: 8 [vl of fHbp]

VAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT YGNGDSLNTG

KLKNDKVSRF DFIRGÎEVOG OUTLESGEF QVYKQSHSAL TAFQTEQÎQD SEHSGKMVAK

RQFRiGDiAG EHTSFDKLPE GGRATYRGTA FGSDDAGGKL TYTIDFAAKO GNGKiEHLKS

PELNVDLAAA DikPDGKRHA ViSGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGi RHIGLAAKO> SEQ ID NO: 9 [v3 of fHbp]

VAADIGTGLADALTAPLDHKDkGLkSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKL

KNDKiSRFDFVOKiEVDGQTfnASGEFQÎYKQNHSAWALQiEKINNPDKTDSLINQRSFLVSGI,

GGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVEI..AAAEL

KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAOEîAGSATVKlGEkVHEIGIAGKQ

B E2017 / 5855> SEQ ID NO: 10 [Pfs25]

KVTVDTVCKR GFUQMSGHt. ECKGENDLVL VNEETCEEKV LKCDEKTVNK PCGDFSKCIK

IDGMPVSYAC KCNLGYDMVN NVGIPNEGKQ VTCGNGKCJL DT8NPVKTGV CSCN1GKVPN VÖDONKCSKD GETKCSLKCL KEQETGKAVO GIYKÙDCKDG FHDGESSiC T> SEQ ID NO: 11 [ROD

AERSTSENRNKRIGGPKLRGNVT8NIKFP.SDNKGKIIRGSNDKLNKNSEDVLEOSEKSLVSENV

PSGLDlDDÎPKESiFIQEDQEGQTHSELNPETSEHSKDLNNNGSKNESSDHSENNKSNKVQNHF

ESLSDl.El, .LENSSaDNL.DKDT! STEPFPNQKHKDLQQDLNDEPLEPFPTQÎHKDYKEKNUNEED

SEPFPRQKHKKVDNHNEEKNWHENGSANGNQGSLKLKSFDEHLKDEKIENÉPIVHENLSIPN

DPiEQiLNQPEQETNIQEQLYNEKQNVEEKQNSQlPSLDLKËPTNEÖILPNHNPLENlKQSESEiN

HVÔÙHALPKENHDKLDNQKËHIDQSQHNINVLQENNINNHQLEPÔEKPNIESFEPKNtDSEIîLPE

NVETEËI! ODVPSPKHSNHETF.EEETSESEHEEAVSEKNA "ETVEHEETVSQESNPEKADNDG

NVSQNSNNELNENÉFVESEKSÉHEARSKPKYEKKVIHGCNFSSNVSSKHTFTDSLDISLVDDSA

HISCNVHLSÉPKYNHLVGLNCPGDIIPDCFFQVYQPÉSEEtEPSIMIVYLDSQINIGDIEYYEDAEG

DOK1KLFGIVGS1PKTTSFTCICKKDKKSAYMTVTIDSARSHHHHHH> SEQ ID NO: 12 [CSPj

MLFQEYQGYGSSSNTRVLNEt.NYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDG

NNNNGDNGREGKDEDKRDGNNEDNEKLRKPKHKKLKQPGDGNPDPNANPNVDPNANPNVD PNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANP nanpnanpnvdpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpnanpn anpnanpnanpnanpnanpnanpnanpnknnqgngqghmmpndpnrnvdenànanimâvkn NNNEEPSDKHlEKYLKKiQNSLSTEY / SPCSVTCGNGiQVRIKPGSANKPKDELDYENDiEKKiCK MEKCSSVFNVVNSSIGLILEHHHHHH> SEQ ID NO; 13 [(NANP) 3]

NÂNPNANPNANP> SEQ ID NO: 14 [CTF1232]

QDQRYISIRNTDTIWLPGNiCAYQFRLDNGGNOEGFGPLTITLaLKDKYGQTLVTRKMETEAFG dsnatrîtdafletecvenvattejïkateesnghrvslplsvfdpqdyhplutvsgknvmleh HHHHH

Claims (15)

1. Immunogenic conjugate comprising a native external membrane vesicle (nOMV), comprising at least one surface saccharide fraction connected to at least one antigen. 2. Immunogenic conjugate according to claim 1, comprising a nOMV comprising at least one surface saccharide fraction connected to a first antigen, in which said first antigen is connected to a different second antigen. 3. Immunogenic conjugate according to claim 1, comprising a nOMV comprising at least one surface saccharide fraction connected to a first antigen, and at least one other surface saccharide fraction connected to a different second antigen. 4. Immunogenic conjugate according to claims 1 to 3, wherein said nOMV is obtained by a detergent-free process, being released into the fermentation broth and purified using centrifugation and subsequent filtration; or being released into the fermentation broth and purified using two consecutive stages of tangential flow filtration (FFT). 5. Immunogenic conjugate according to the preceding claims, in which said nOMV is produced from wild type bacteria or from genetically modified bacterial strains which are mutated to amplify the production of vesicles, and possibly also to eliminate or modify antigens and / or to overexpress antigens BE2017 / 5855 homologs or antigens from other organisms. 6. Immunogenic conjugate according to the preceding claims, in which said nOMV is obtained from a bacterium chosen from: Neisseria, Shigella, serovars of Salmonella enterica, Haemophilus influenzae, Vibrio cholerae, Bordetella pertussis, Mycobacterium smegmatis, Mycobacterium bovis BCG, Escherichia coli, Bacteroides, Pseudomonas aeruginosa, Helicobacter pylori, Brucella melitensis Campylobacter jejuni, Actinobacillus actinomycetemcomitans, Xenorhabdus nematophilus, Moraxella catarrhalis, or Borrelia burgdorferi. 7. Immunogenic conjugate according to the preceding claims, wherein the antigen is an immunogenic polypeptide or a capsular polysaccharide. 8. Immunogenic conjugate according to the preceding claims, wherein said nOMV and the antigen are derived from the same bacterial strain or from different bacterial strains. 9. Immunogenic conjugate according to the preceding claims, in which said nOMV and at least one antigen are chosen as follows: NOMVs Selected antigen Salmonella typhimurium Neisseria meningitidis fHbp Salmonella typhimurium CSP of Plasmodium falciparum Salmonella typhimurium Pfs25 of Plasmodium falciparum Salmonella typhimurium RO6C of Plasmodium falciparum BE2017 / 5855 Salmonella typhimurium ROIOC of Plasmodium falciparum Salmonella typhimurium CTF1232 of Escherichia coli Salmonella typhimurium Saccharide Vi from S. typhimurium Neisseria meningitidis Neisseria meningitidis fHbp Neisseria meningitidis Poly- oligosaccharide rhamnose Shigella CTF1232 of Escherichia coli Neisseria meningitidis B Capsular saccharide from MenA Neisseria meningitidis B Capsular saccharide from MenC 10. Immunogenic conjugate according to claim 9, in which said nOMV and at least one antigen are chosen as follows: NOMVs Selected antigen Neisseria meningitidis B Capsular saccharide from MenA Neisseria meningitidis B Capsular saccharide from MenC 11. The immunogenic conjugate according to claim 2, wherein said first antigen is Pfs25, and said second antigen is (NANP) 3. 12. Immunogenic conjugate according to claim 11, wherein the nOMV is obtained from Salmonella typhimurium. BE2017 / 5855 13. The immunogenic conjugate according to claim 2, wherein said first antigen is a capsular saccharide from MenA, and said second antigen is a capsular saccharide from MenC. 14. Immunogenic conjugate according to claim 13, in which the nOMV is obtained from a strain of Meningococcus of serogroup B, preferably expressing the vl and 2 of fHbp. 15. Immunogenic conjugate according to claims 1 to 14, wherein the saccharide fraction of nOMV and the at least one antigen are connected together via a bivalent linker. 16. An immunogenic conjugate according to the preceding claims, wherein said nOMV is a GMMA vesicle. 17. Process for the preparation of the immunogenic conjugate according to claims 1 to 16, comprising the following steps: i) the activation of at least one surface saccharide fraction of nOMV, and ii) the connection of the activated saccharide thus obtained to at least one antigen, optionally via a bivalent linker. 18. The method of claim 17, wherein the activation step i) comprises the oxidation of one or more hydroxyl groups of said saccharide fraction of said nOMV in aldehyde functionality. 19. Method according to any one of claims 17 or 18 in the presence of an alkaline bisulfite. BE2017 / 5855 20. Immunogenic composition comprising an immunogenic conjugate according to claims 1 to 16 and at least one pharmaceutically acceptable carrier or excipient. 21. A conjugate or immunogenic composition according to any one of claims 1 to 16 or 20, for use as a medicament. 22. A conjugate or immunogenic composition for use according to claim 21 for inducing an immune response in a vertebrate. 10 23. A vaccine comprising the conjugate or the immunogenic composition according to any one of claims 1 to 16 or 20. 24. Use of nOMV for the preparation of immunogenic conjugates. 25. Use of nOMV according to claim 24, wherein said immunogenic conjugates are as defined in any one of claims 1 to 16.